(Pharmacology-Research, Safety Testing and Regulation) Paolo Fubini-Mannitol - Chemistry, Uses and Potential Side Effects-Nova Science Pub Inc (2013)

PHARMACOLOGY - RESEARCH, SAFETY TESTING AND REGULATION
MANNITOL
CHEMISTRY, USES AND
POTENTIAL SIDE EFFECTS
No part of this digital document may be reproduced, stored in a retrieval system or transmitted in any form or
by any means. The publisher has taken reasonable care in the preparation of this digital document, but makes no
expressed or implied warranty of any kind and assumes no responsibility for any errors or omissions. No
liability is assumed for incidental or consequential damages in connection with or arising out of information
contained herein. This digital document is sold with the clear understanding that the publisher is not engaged in
rendering legal, medical or any other professional services.
PHARMACOLOGY - RESEARCH, SAFETY
TESTING AND REGULATION
Additional books in this series can be found on Novas website

under the Series tab.
Additional e-books in this series can be found on Novas website

under the e-book tab.
BIOTECHNOLOGY IN AGRICULTURE,
INDUSTRY AND MEDICINE
Additional books in this series can be found on Novas website

under the Series tab.
Additional e-books in this series can be found on Novas website

under the e-book tab.
PHARMACOLOGY - RESEARCH, SAFETY TESTING AND REGULATION
MANNITOL
CHEMISTRY, USES AND
POTENTIAL SIDE EFFECTS
PAOLO FUBINI
EDITOR
New York
Copyright 2013 by Nova Science Publishers, Inc.
All rights reserved. No part of this book may be reproduced, stored in a retrieval system or
transmitted in any form or by any means: electronic, electrostatic, magnetic, tape,
mechanical photocopying, recording or otherwise without the written permission of the
Publisher.
For permission to use material from this book please contact us:
Telephone 631-231-7269; Fax 631-231-8175
Web Site: http://www.novapublishers.com
NOTICE TO THE READER

The Publisher has taken reasonable care in the preparation of this book, but makes no
expressed or implied warranty of any kind and assumes no responsibility for any errors or
omissions. No liability is assumed for incidental or consequential damages in connection
with or arising out of information contained in this book. The Publisher shall not be liable
for any special, consequential, or exemplary damages resulting, in whole or in part, from
the readers use of, or reliance upon, this material. Any parts of this book based on
government reports are so indicated and copyright is claimed for those parts to the extent
applicable to compilations of such works.
Independent verification should be sought for any data, advice or recommendations

contained in this book. In addition, no responsibility is assumed by the publisher for any
injury and/or damage to persons or property arising from any methods, products,
instructions, ideas or otherwise contained in this publication.
This publication is designed to provide accurate and authoritative information with regard
to the subject matter covered herein. It is sold with the clear understanding that the
Publisher is not engaged in rendering legal or any other professional services. If legal or any
other expert assistance is required, the services of a competent person should be sought.
FROM A DECLARATION OF PARTICIPANTS JOINTLY ADOPTED BY A
COMMITTEE OF THE AMERICAN BAR ASSOCIATION AND A COMMITTEE OF
PUBLISHERS.
Additional color graphics may be available in the e-book version of this book.
Library of Congress Cataloging-in-Publication Data

ISBN: (eBook)
Library of Congress Control Number: 2013945783
Published by Nova Science Publishers, Inc. New York

CONTENTS
Preface vii
Chapter 1 Utilization and Production of D-Mannitol by Bacteria 1
Josef Deutscher, Houda Bouraoui, Meriem Derkaoui
and Philippe Joyet
Chapter 2 Concentration of Mannitol and Other Soluble
Carbohydrates in the Crustose Lichen Rhizocarpon
Geographicum 21
Richard A. Armstrong
Chapter 3 Mannitol: Disease-Related Changes and their Use
for Clinical Disorders 41
David Caldern Guzmn, Gerardo Barragn Meja,
Hugo Jurez Olgun and Ernestina Hernndez Garca
Chapter 4 Use of Mannitol in Thermal Energy Storage Applications 63
Luisa F. Cabeza, Camila Barreneche, Antoni Gil
and A. Ins Fernndez
Chapter 5 Chiral Phosphorous Ligands Derived from D-Mannitol:
Synthesis and their Applications in Asymmetric Catalysis 87
Yingwei Zhao, Lei Yang and Hanmin Huang
Index 113
PREFACE
In this book, the authors present topical research in the study of the
chemistry, uses and potential side effects of mannitol. Topics discussed
include the utilization and production of D-mannitol by bacteria; concentration
of mannitol and other soluble carbohydrates in the crustose lichen rhizocarpon
geographicum; disease-related changes and mannitols use for clinical
disorders; use of mannitol in thermal energy storage applications; and chiral
phosphorous ligands derived from D-mannitol.
Chapter 1 - Certain plants, yeasts, algae, lichen and fungi produce large
amounts of D-mannitol and many bacteria developed the capacity to utilize
this naturally occurring carbon and energy source. For that purpose they use
different transport systems and catabolic pathways. Some bacteria transport D-
mannitol via ion-driven co-transport systems or ABC transporters without
modification of their substrate. Intracellular D-mannitol is subsequently
converted into fructose by the D-mannitol 2-dehydrogenase MtlD (l) and
fructose is phosphorylated to the glycolytic intermediate fructose-6-P.
Numerous other bacteria take up D-mannitol via the phosphoenolpyruvate
(PEP): carbohydrate phosphotransferase system (PTS), which catalyzes the
transport and concomitant phosphorylation of its substrates. D-mannitol
transported by the PTS therefore arrives as D-mannitol-1-P in bacterial cells.
The enzyme D-mannitol-1-P 5-dehydrogenase MtlD(s) converts D-mannitol-
1-P into fructose-6-P. The genes encoding the different types of transport and
catabolic enzymes are usually organized within the mtl operon. Transcription
of the mtl operon is also controlled by various mechanisms, which allow the
expression of the mtl genes only when mannitol is present in the growth
medium. The mtl operon is usually also submitted to carbon catabolite
repression, which prevents its expression when an efficiently metabolizable
viii Paolo Fubini
carbon source, such as glucose, is present. Bacteria not only utilize mannitol,
but some are also able to produce D-mannitol from fructose with the aid of the
enzyme Mdh. This is of biotechnological interest and improvement of the
yield of mannitol biosynthesis might allow a cheaper and more efficient
production of D-mannitol compared to the industrial production via catalytic
reduction of fructose.
Chapter 2 - In symbiotic lichens which have Trebouxia as the algal
partner, photosynthesis by the algae results in the production of the soluble
carbohydrate ribitol which is then transported to the fungus where it is
converted to arabitol and mannitol. Within the fungus, arabitol may act as a
short-term carbohydrate reserve while mannitol may have a more protective
function and be important in stress resistance. The concentrations of ribitol,
arabitol, and mannitol were measured, using gas chromatography, in the
central areolae and marginal hypothallus of the crustose lichen Rhizocarpon
geographicum (L.) DC. growing on slate rocks in north Wales, UK. The
concentrations of all three soluble carbohydrates were greater in the central
areolae than in the marginal prothallus. In addition, the ratio of mannitol in the
prothallus to that in the areolae was least in July. The concentration of an
individual carbohydrate in the prothallus was correlated primarily with the
concentrations of the other carbohydrates in the prothallus and not to their
concentrations in the areolae. Low concentration of ribitol, arabitol, and
mannitol in the marginal prothallus compared with the central areolae suggests
either a lower demand for carbohydrate by the prothallus or limited transport
from areolae to prothallus and may explain the low growth rates of this
species. In addition, soluble carbohydrates appear to be partitioned differently
through the year with an increase in mannitol compared with arabitol in more
stressful periods.
Chapter 3 - Mannitol, a white, crystalline alcohol, is derived from sugar
by reduction. The pathway to obtain mannitol from natural products is through
hydrogenation of fructose, which is formed from either starch or sugar. This
substance is present in a wide variety of natural products, and in almost all
plants. It is used as an osmotic diuretic agent and a weak renal vasodilator.
Aqueous solutions of mannitol are mildly acidic and sometimes such solutions
are used to decrease the pH. Mannitol is clinically used in osmotherapy to
temporarily reduce acute intracranial pressure while waiting for the definitive
treatment. It is also used to treat patients with oliguric renal failure.
Consequently, mannitol increases water and Na+ excretion, thereby decreasing
extracellular fluid volume. It can also be used as a facilitating agent for
transporting drug agents directly into the brain. This chapter reviews the
Preface ix
possible mechanisms of mannitol and its metabolite that are involved in

common clinical disorders. Moreover, the analytical techniques and
biochemical markers used to monitor mannitol and its metabolite are described
in this recompilation.
Chapter 4 - Nowadays thermal energy storage (TES) systems are proposed
as one of the most powerful technologies to be charged with heat (or cold) and
hold energy over time by shifting demand over time to reduce peak loads and
facilitating the greater use of renewable energy by storing the energy produced
so it can coincide with demand. TES systems are able to store energy as
sensible heat leading with temperature increment of the storage medium, as
latent heat storing energy using the latent heat produced when a phase change
state occur using phase change materialsPCM, and as chemical reaction
energy storing energy using a exothermic/endothermic reversible reaction by
thermochemical materials (TCM). Solar cooling and air-conditioning is a
technology that allows coincidence of solar gains with cooling loads reducing
peak loads created by air-conditioning. TES systems can be coupled between
absorption chillers and solar collectors in order to use the energy stored when
the there is a peak load or the system is practically discharged. In addition,
phase change materials PCM candidates must fulfill several conditions to be
used as storage materials: melting point of PCM must be closed to selected
work temperature range, high latent heat and high specific heat, elevate
thermal conductivity (solid and liquid state) to support charging and
discharging processes inside the storage system. Additionally, the change
volume during phase change transformation must be minimum, as well as the
pressure vapor, allowing the use of conventional containers. Moreover, it must
melt congruently with minimum subcooling and it must be chemically stable.
D-mannitol has a phase change temperature at 167 C and a phase change
enthalpy is around 316 kJkg-1. These thermophysical properties turn d-
mannitol as a perfect candidate to be used as PCM and it was studied with this
purpose. D-mannitol was characterized performing differential scanning
calorimetry under dynamic mode using a 0.5 Kmin-1 heating rate between 25
C and 200 C. This substance was cycled several times and results shows 3
different thermal behavior: The first one was a single peak at 167 C, the
second has double peak at 156 C and 167 C, and the third thermal behavior is
a single peak at 157 C. Accordingly, there is a polymorphic transformation
which was studied with FT-IR and two different phases were identified: -
phase and -phase. Then, a temperature range was established to work with
this substance as PCM between 135 C and 175 C. This working range
includes the phase change transformation of the two phases under analysis (
x Paolo Fubini
and ). To test the d-mannitol thermal behaviour at pilot plant scale, 150 kg of
d-mannitol were introduced in a storage tank which was designed as shell-and-
tubes heat exchanger. Results show that applying different cooling conditions
produces d-mannitol polymorphic changes. Moreover, it has been shown that
the working range (between 135C and 175C) is adequate for pilot plant
experiments.
Chapter 5 - As a kind of readily available carbohydrate, D-mannitol has
been widely used in the synthesis of chiral phosphorous ligands. This chapter
reviews the design and synthesis of various mono- and diphosphorous ligands
based on D-mannitol backbone. Also, the successful applications of these
ligands in asymmetric catalysis, such as enantioselective hydrogenation and
enantioselective conjugate addition, are summarized.
In: Mannitol ISBN: 978-1-62808-762-8
Editor: Paolo Fubini 2013 Nova Science Publishers, Inc.
Chapter 1
UTILIZATION AND PRODUCTION

OF D-MANNITOL BY BACTERIA
Josef Deutscher1,2,3,*, Houda Bouraoui1,2,4,

Meriem Derkaoui1,2 and Philippe Joyet1,2
1
INRA, Microbiologie de l'alimentation au service de la sant humaine
(MICALIS), UMR1319, 78350 Jouy en Josas, France
2
AgroParisTech, MICALIS, 78350 Jouy en Josas, France
3
CNRS, MICALIS, SNC9130, F-78350 Jouy en Josas, France
4
Dpartement de Biologie, Universit de Batna, Algrie
ABSTRACT
Certain plants, yeasts, algae, lichen and fungi produce large amounts of
D-mannitol and many bacteria developed the capacity to utilize this naturally
occurring carbon and energy source. For that purpose they use different
transport systems and catabolic pathways. Some bacteria transport D-
mannitol via ion-driven co-transport systems or ABC transporters without
modification of their substrate. Intracellular D-mannitol is subsequently
converted into fructose by the D-mannitol 2-dehydrogenase MtlD (l) and
fructose is phosphorylated to the glycolytic intermediate fructose-6-P.
Numerous other bacteria take up D-mannitol via the phosphoenolpyruvate
(PEP): carbohydrate phosphotransferase system (PTS), which catalyzes the
transport and concomitant phosphorylation of its substrates. D-mannitol
*
Corresponding author: Josef.Deutscher@grignon.inra.fr.
2 Josef Deutscher, Houda Bouraoui, Meriem Derkaoui et al.
transported by the PTS therefore arrives as D-mannitol-1-P in bacterial cells.

The enzyme D-mannitol-1-P 5-dehydrogenase MtlD(s) converts D-mannitol-
1-P into fructose-6-P. The genes encoding the different types of transport and
catabolic enzymes are usually organized within the mtl operon. Transcription
of the mtl operon is also controlled by various mechanisms, which allow the
expression of the mtl genes only when mannitol is present in the growth
medium. The mtl operon is usually also submitted to carbon catabolite
repression, which prevents its expression when an efficiently metabolizable
carbon source, such as glucose, is present. Bacteria not only utilize mannitol,
but some are also able to produce D-mannitol from fructose with the aid of
the enzyme Mdh. This is of biotechnological interest and improvement of the
yield of mannitol biosynthesis might allow a cheaper and more efficient
production of D-mannitol compared to the industrial production via catalytic
reduction of fructose.
INTRODUCTION
The hexitol D-mannitol is by far the most abundant sugar alcohol in nature.
This sugar alcohol has the same stereochemical configuration as mannose and
therefore possesses a two-fold symmetry axis. Rotation of the hexitol by 180
provides a molecule with identical configuration and the 1- and 6-position are
therefore indistinguishable. Nevertheless, phosphorylated mannitol is usually
referred to as mannitol-1-P and not as mannitol-6-P. D-Mannitol is produced in
relatively large quantities for example by certain marine algae. In the brown
seaweed Laminaria japonica mannitol is the most abundant carbohydrate.
Enterobacter sp. JMP3 was recently shown to efficiently convert D-manitol
produced by the seaweed into bioethanol [1]. In algae the polyol exerts multiple
functions, such as osmoregulation, storage, regeneration of reducing power, and
scavenging of active oxygen species [2, 3]. In these organisms, D-mannitol is
mainly produced from fructose-6-P, which is reduced to D-mannitol-1-P by the
enzyme D-mannitol-1-P 5-dehydrogenase and subsequently dephosphorylated to
D-mannitol [4]. When D-mannitol-producing algae are transferred from a saline
medium (sea water) to fresh water most of the accumulated intracellular D-
mannitol is released into the environment by a yet unknown efflux system [5, 6].
In higher vascular plants D-mannitol is also one of the major photosynthetic
products [7] and also protects against osmotic pressure and stress [2]. In celery
and some related plants biosynthesis does not seem to occur from fructose-6-P but
from mannose-6-P, which is reduced by an NADPH-dependent mannose-6-P 1-
reductase to D-mannitol-1-P [8, 9]. Finally, a D-mannitol-1-P-specific
phosphatase dephosphorylates D-mannitol-1-P to D-mannitol [6, 10]. Some plants
Utilization and Production of D-Mannitol by Bacteria 3
contain in the roots a mannose 1-oxidoreductase, which converts D-mannitol into

mannose and not fructose [11]. The resulting mannose is specifically used for
growth of the roots. Similar to algae, fungi and mushrooms primarily use an
NADH-dependent D-mannitol-1-P 5-dehydrogenase to convert fructose-6-P into
mannitol-1-P. Similar as in plants, an acid phosphatase was identified in
mushrooms that catalyzes the dephosphorylation of D-mannitol-1-P to D-
mannitol [12]. To a lesser extent fungi and mushrooms produce D-mannitol also
from fructose-6-P by the NADH-dependent D-mannitol dehydrogenase [10].
Several bacteria, mostly heterofermentative lactic acid bacteria, are also able
to produce D-mannitol when grown on specific media [13] and this
biotechnological aspect will be discussed in detail. However, seen the abundance
of D-mannitol in nature it is not surprising that most bacteria utilize the hexitol as
a carbon and energy source. In this chapter we will describe the different bacterial
transport systems used for the uptake of D-mannitol as well as the various
catabolic routes and the enzymes catalyzing its catabolism. The synthesis of the
enzymes required for the transport and metabolism of D-mannitol is tightly
regulated. We will also describe the various mechanisms controlling induction
and carbon catabolite repression of the various types of mtl operons.
UPTAKE AND METABOLISM OF D-MANNITOL

Most bacteria do not produce D-mannitol but use it as a carbon and energy
source. For that purpose they developed transport systems allowing the uptake of
the hexitol from the environment. Three major types of bacterial D-mannitol-
specific transport systems can be distinguished: Uptake by ion gradient-driven
transporters belonging to the major facilitator superfamily, uptake by ATP
binding cassette (ABC) transporters and uptake via the phosphoenolpyruvate
(PEP):carbohydrate phosphotransferase system (PTS) (Figure 1). Facilitated
diffusion of D-mannitol similar to that reported for the triol glycerol has so far not
been reported. Uptake via ion gradient-driven transporters or ABC transporters
occurs without any modification of the substrate. In contrast, during transport via
the PTS D-mannitol is phosphorylated at the 1-position and therefore arrives as
D-mannitol-1-P in the cytoplasm of bacterial cells. In bacteria taking up D-
mannitol via an ion gradient-driven transporter (MtlT) or an ABC transport
system (MtlEFGK) intracellular D-mannitol is usually first oxidized to D-fructose
by the enzyme D-mannitol 2-dehydrogenase. The genes for the dehydrogenase
and the ion gradient-driven transporter are organized in the mtlTD operon (Figure
2) [14]. This is also true for the genes encoding the D-mannitol ABC transporter
of the family pseudomonadaceae, where the mtl operon is formed by the

mtlEFGKDZY genes (Figure 2), with mtlZ and mtlY encoding carbohydrate
kinases. The MtlD proteins of corynebacteria and pseudomonadaceae have a
similar length (about 500 amino acids) and exhibit significant sequence identity
(more than 40%). They belong to the long chain alcohol dehydrogenases [15]. The
ABC transporter of Pseudomonas fluorescens has a relatively broad substrate
specificity and in addition to D-mannitol takes up also D-glucitol and arabitol
[16]. Consequently, the dehydrogenase MtlD (l) of the members of this family
oxidizes the two hexitols to fructose and the pentitol arabitol to xylulose [17].
Figure 1. Schematic presentation of the three different known D-mannitol uptake systems,
the catabolic pathways of D-mannitol and a potential D-mannitol efflux system present in
some heterofermentative lactic acid bacteria. D-Mannitol can be transported by bacteria
via ion-driven permeases (MtlT), ABC transport systems (MtlEFGK) or PTS permeases
(MtlA or MtlA and MtlF). D-Mannitol transported by the PTS arrives as D-mannitol-1-P
in the cell and is subsequently converted to the glycolytic intermediate D-fructose-6-P by a
short-chain alcohol dehydrogenase MtlD(s). D-Mannitol taken up by an ion-driven
permease or an ABC transport system is first oxidized to D-fructose by a long-chain
alcohol dehydrogenase MtlD (l) and subsequently phosphorylated to D-fructose-6-P. MtlE
of the ABC transport complex is a D-mannitol binding protein located in the periplasm,
MtlK is an ATP hydrolyzing protein providing the energy for the transport process and
MtlF and MtlG are two membrane-spanning proteins. Some heterofermentative lactic acid
bacteria produce D-mannitol from D-fructose in an NADPH-requiring reaction catalyzed
by the enzyme Mdh, another type of mannitol 2-dehydrogenase. D-Mannitol might be
secreted into the medium via an ABC efflux system, the genes of which are frequently
located in the vicinity of the mdh gene.
Figure 2. The gene organization of D-mannitol-specific regions in different bacteria.

Shown are the D-mannitol-specific regions for organisms transporting D-mannitol via the
PTS, ABC transport systems or ion-driven permeases. The genes encoding D-mannitol or
D-mannitol-1-P dehydrogenases (both called mtlD) belonging either to the long- or the
short-chain alcohol dehydrogenases, respectively, are distinguished by adding (l) or (s) to
mtlD. The tetR gene encodes a TetR-like and deoR a DeoR-like transcription activator and
reg an unknow regulator. The mdh gene codes for an NADP+-dependent D-mannitol
dehydrogenase, fruK as well as mtlZ for a fructokinase, mtlY for a xylulose kinase, and
mtlT for an ion-driven D-mannitol permease of the major facilitator superfamily. The
D-mannitol-specific ABC transport system of pseudomonodaceae is encoded by mtlE
(D-mannitol binding protein), mtlK (ATP binding protein) and mtlF and mtlG (two trans-
membrane transport proteins). The transcription activator MtlR has been identified for
strain DSM 50106 [42] and a similar gene is located upstream from mtlE in strain SBW25,
in others quite distant from the mtl operon. The organization of D-mannitol PTS regions is
highly variable and the examples presented here are not exhaustive. The most common
operon organization in enterobacteriaceae is mtlADR and in firmicutes mtlARFD. Highly
remarkable are the co-localization of the ptsHI (ptsIH) genes encoding the general PTS
proteins EI and HPr with the D-mannitol-specific PTS genes in L. xyli and A. arilaitensis,
the duplication of the EIIB domain in several homofermentative lactic acid bacteria and
the duplication of all D-mannitol-specific PTS domains/proteins in several enterococci.
Corynebacterium glutamicum was reported to lack a fructokinase converting

fructose into fructose-6-P and fructose formed from D-mannitol was therefore
found to be secreted into the medium and subsequently taken up and
phosphorylated by a fructose-specific PTS [14]. One of the carbohydrate kinases
encoded by the mtl operon, MtlZ, strongly resembles fructokinase from Vibrio
alginolyticus and has indeed been shown to convert fructose formed from D-
mannitol or D-glucitol by the D-mannitol dehydrogenase MtlD(l) into fructose-6-
P [16]. The other kinase MtlY phosphorylates xylulose formed by MtlD(l) from
arabitol into xylulose-5-P [16].
Gram-positive organisms including firmicutes and actinobacteria as well as
enterobacteriaceae take up D-mannitol via a PTS and phosphorylate it already
during its transport to D-mannitol-1-P, which is subsequently converted to
fructose-6-P by the enzyme D-mannitol-1-P 5-dehydrogenase (MtlD).
Unfortunately, the nomenclature is ambiguous because both, the D-mannitol 2-
dehydrogenase of bacteria transporting D-mannitol via ion-driven transporters or
ABC transport systems as well as D-mannitol-1-P 5-dehydrogenase of bacteria
transporting D-mannitol via a PTS were called MtlD. D-Mannitol-1-P 5-
dehydrogenases and D-mannitol 2-dehydrogenase have a different length (about
370 and 500 amino acids, respectively) and belong to the short-chain and long-
chain alcohol dehydrogenases [15]. In this article they will be distinguished as
MtlD(l) for the long-chain and MtlD(s) for the short-chain alcohol
dehydrogenases. The two types of alcohol dehydrogenases probably have the
same evolutionary origin, because despite their different lengths the C-terminal
part of mannitol-1-P 5-dehydrogenases from firmicutes and enterobacteriaceae
exhibits significant sequence similarity (about 40%) to the central part of mannitol
dehydrogenases from corynebacteria and pseudomonadaceae.
In order to phosphorylate its substrate during the transport step four of the
ususally five PTS components form a phosphorylation cascade (Figure 3).
Enzyme I (EI) autophosphorylates with PEP and transfers the phosphoryl group to
the histidyl residue of the second general PTS protein HPr. P~His-HPr
phosphorylates one of usually several sugar-specific EIIA components and
P~EIIA donates its phosphoryl group to the cognate EIIB. In the last step, P~EIIB
phosphorylates the carbohydrate (sugar, sugar alcohol, amino sugar and many
other sugar derivatives) bound to the corresponding membrane integral EIIC.
Phosphorylation of the carbohydrate lowers the affinity for its EIIC and the
phosphorylated substrate is released into the cytoplasm (Figure 3) [18]. The EII
components are frequently fused together providing one single protein. This is the
case for the D-mannitol-specific MtlA protein of E. coli and other
enterobacteriaceae, which is composed in the following order of the EIIC, EIIB
and EIIA domains. Several actinobacteria, such as Arthrobacter aurescens, also
possess an EIICBAMtl protein composed of the three mannitol-specific domains.
This is also true for Corynebacterium durum, while, as mentioned above, most
other corynebacteria possess an ion-driven transporter MtlT [14]. In contrast, in
other actinobacteria, such as Leifsonia xyli and Nakamurella multipartita as well

as in all firmicutes studied so far the D-mannitol-specific EIIA component
(EIIAMtl) is a distinct protein encoded by the mtlF gene and only the EIIBMtl
domain is fused to the membrane-spanning EIICMtl (Figure 3) [19, 20].
Figure 3. Schematic presentation of the different modes of organization of D-mannitol-

specific PTSs. In all organisms transporting D-mannitol via a PTS, the general PTS
proteins EI and HPr catalyze the PEP dependent phosphorylation of the D-mannitol-
specific PTS proteins. In E. coli, the D-mannitol-specific PTS components EIIAMtl, EIIBMtl
and EIICMtl are fused to a single protein called MtlA. The EIIAMtl and EIIB Mtl domains are
therefore located at the inner side of the cytoplasmic membrane. In firmicutes, such as B.
subtilis, only the EIIBMtl and EIICMtl domains are fused together to form MtlA, whereas
EIIAMtl exists as a distinct soluble, cytoplasmic protein. In both types of organisms, P~His-
HPr phosphorylates a conserved histidyl residue in EIIA Mtl and P~EIIAMtl transfers the
phosphoryl group to a cysteine residue located in the N-terminus of the EIIBMtl domain. In
the last step, P~EIIBMtl donates its phosphoryl group to a D-mannitol molecule bound to
the membran-spanning EIICMtl domain. Phosphorylation of D-mannitol lowers its affinity
for EIICMtl and the phosphorylated sugar alcohol is released into the cytoplasm. Shown are
also the mannitol-specific PTS components of L. casei. In this organism as well as in
several other homofermentative lactic acid bacteria, MtlA contains two EIIBMtl domains. It
is not clear whether P~EIIAMtl transfers its phosphoryl group only to one or to both EIIB Mtl
domains and, if the latter possibility is correct, whether both P~EIIBMtl domains can
phosphorylate D-mannitol bound to the EIICMtl domain.
Interestingly, some firmicutes, such as Streptococcus mutans [21] or

Lactobacillus casei [22], contain two EIIBMtl domains fused to EIICMtl. In several
enterococci, such as E. faecalis, Enterococcus faecium, Enterococcus

casseliflavus, etc., all three PTS domains are duplicated (Figure 2) [23]. In these
enterococci the gene order is mtlA1RF1A2F2D, with frequently one or two ORFs
oriented in the opposite direction inserted between mtlF1 and mtlA2. One possible
explanation for the presence of two PTS might be that one functions as mannitol
sensor by controlling the activity of MtlR and this PTS might have only low
mannitol transport activity, whereas the other PTS functions as the main mannitol
transporter. A similar partition of transport and regulatory functions has been
reported for the two glucose/mannose PTS transporters of Listeria monocytogenes
[24]. Also noteworthy, in actinobacteria the genes ptsHI (or ptsIH, gene order
sometimes inversed compared to enterobacteriaceae and firmicutes) encoding EI
and HPr are frequently located upstream from the mannitol operon either oriented
in the same (L. xyli) or in the opposite direction (A. aurescens) (Figure 2).
The EIIAMtl and EIIBMtl proteins or domains from various organisms have
been purified to homogeneity and the PEP-requiring phosphorylation cascade
including EI and HPr has been reconstituted in vitro [23, 25-27]. It is noteworthy
that the E. coli EIIBMtl domain was the first PTS component shown to be
phosphorylated at a cysteine residue [28]. The structural changes induced by the
phosphorylation of the E. coli EIIAMtl and EIIBMtl components have been studied
by NMR spectroscopy. NMR spectroscopy was also used to characterize the
interaction complex of the EIIAMtl and EIIBMtl domains of E. coli [29] as well as
the interaction complex of EIIAMtl with HPr [30]. The entire E. coli D-mannitol-
specific PTS permease MtlA was also purified to homogeneity and has been
extensively studied [31, 32]. It is a dimeric protein [33] with each subunit of the
homo-oligomer containing probably seven transmembrane helices [34].
Preliminary information about the D-mannitol binding site in the EIICMtl
component was obtained by carrying out FRET (Frster resonance energy
transfer) experiments [35]. The mtlD gene encoding D-mannitol-1-P 5-
dehydrogenase, MtlD(s), is usually organized in an operon together with the
gene(s) coding for the D-mannitol-specific EII PTS components. In addition,
located upstream from or in firmicutes also frequently within the D-mannitol
operon is the gene encoding the transcription regulator usually called MtlR, which
depending on the organism can be a repressor or an activator. The gene order for
the D-mannitol operon in firmicutes is therefore often mtlARFD. Bacillus subtilis
is an exception to this rule, because the transcription activator of its D-mannitol
operon is located about 14.5 kb downstream from it [36] (Figure 2). In
Enterococcus mundtii a gene encoding an MtlR-like protein is located about 13 kb
upstream from the mtl operon, which in the organisms containing an isolated mtlR
has the gene order mtlAFD.
REGULATION OF MTL OPERON EXPRESSION

Bacteria have developed sophisticated regulatory mechanisms allowing them
to express catabolic genes and operons only when the corresponding substrate is
present in the environment. This type of regulation is called catabolic induction
and depending on the proteins involved in it several different mechanisms exist.
Again, the nomenclature is quite confusing because independent of whether it is a
repressor or activator mechanism that controls the expression of the mtl operon,
the proteins involved in it are always called MtlR. As a consequence at least four
different types of MtlR proteins exist which exhibit neither functional nor
sequence similarity. The classical mechanism of catabolic induction involves a
repressor protein with usually an N-terminal helix-turn-helix (HTH) DNA binding
motif which allows its interaction with the operator site. The operator site usually
either overlaps the promoter region or is located between promoter and the start
codon. In the first case, the repressor prevents binding of the RNA polymerase
holoenzyme and in the second case it functions as a transcription roadblock. Even
if not included in the mtl operon, MtlR frequently regulates the expression of its
own gene. This is the case for the mtlDT operon of Arthrobacter arilaitensis and
C. glutamicum where the mtlR gene, which encodes a DeoR-like repressor, is
located upstream from the mtlDT operon and oriented in opposite direction [14]
(Figure 2). In this organism, D-mannitol is taken up by MtlT, an ion-driven
transporter. A small portion of the transported D-mannitol probably binds to the
repressor, which causes structural changes preventing its interaction with the
operator sites located in front of mtlDT and mtlR, thus leading to induction of both
transcription units. The increased production of MtlR counteracts the induction
effect and thus allows over a wide range a smooth increase of mtlDT expression in
response to the increase of the extracellular D-mannitol concentration.
The E. coli mtlR gene was originally also thought to encode a repressor for its
mtlADR operon encoding the PTS permease MtlA, the D-mannitol-1-P 5-
dehydrogenase, MtlD(s), and the regulator MtlR (Figure 2). An identical
organisation of the mannitol operon is found in most enterobacteriaceae, as was
shown for Klebsiella pneumoniae [37], but is also found in the order Vibrionales
[38]. Inactivation of the E. coli mtlR gene indeed caused constitutive expression of
mtlADR [39]. However, MtlR did not bind to any of the two presumed operator
sites present in the mtlADR leader region. In addition, solving the crystal structure
of E. coli MtlR revealed that this protein contains no known DNA binding motif
and it was therefore proposed that MtlR would only indirectly control mtlADR
expression [40]. Indeed, it was recently reported that in Vibrio cholerae a small
RNA (MtlS sRNA) transcribed antisense to the 5 untranslated region of the
mtlADR operon controls expression of the mannitol operon of this organism [41].
It is tempting to assume that MtlR regulates the amount of MtlS in response to the
presence or absence of D-mannitol.
The mtlR gene of the -proteobacterium P. fluorescens strain DSM50106
encodes a regulatory protein of about 34 kDa belonging to the AraC/XylS family
of transcription regulators [42], which contain a C-terminal HTH DNA binding
domain [43]. P. fluorescens MtlR was identified as transcription activator, which
when produced in E. coli allowed the mannitol-induced expression of the galK
reporter gene fused to the promoter region of the P. fluorescens mannitol operon.
In the presence of D-mannitol purified MtlR specifically binds to a DNA fragment
containing the promoter/operator region of the mtlEFGKDZY operon (Figure 2),
which encodes the four proteins of an ABC transporter, a D-mannitol 2-
dehydrogenase and two carbohydrate kinases (Figure 2). P. fluorescens MtlR is
probably activated not only by mannitol, but also by glucitol and arabitol, because
as mentioned before these two polyols are also taken up by the ABC transporter
[16]. In P. fluorescens strain SBW25 a gene encoding a protein nearly identical to
MtlR of strain DSM50106 is located just upstream from the mannitol operon
(Figure 2). In other strains (SS101) the mtlR gene is loctaed far away from the mtl
operon.
In firmicutes the expression of the mannitol operon is also controlled by a
transcription activator called MtlR [21, 27, 44, 45]. However, MtlR of firmicutes
is not at all related to MtlR of -proteobacteria. MtlR of firmicutes is composed of
an N-terminal DNA binding domain and an Mga-like domain followed by four
regulatory domains containing potential PTS phosphorylation sites (Figure 4)
[46]. The penultimate regulatory domain resembles EIIBGat and the last one
EIIAMtl PTS components. They are preceded by two domains which were called
PTS regulation domains (PRD). PRDs are found in antiterminators and
transcription activators of firmicutes, actinobacteria and certain proteobacteria
[18]. Each PRD usually contains two conserved histidyl residues potentially
phosphorylated by PTS components. The MtlR transcription activators of
firmicutes therefore possess up to six presumed PTS phosphorylation sites, five of
them being histidines and one being a cysteine. The activity of PRD-containing
transcription regulators is indeed controlled by PTS-catalyzed phosphorylation at
two or sometimes three of the potential phosphorylation sites. Although the
sequence of PRD-containing MtlR proteins is strongly conserved it seems that
their mode of regulation can largely vary. PRD-containing MtlRs are usually
controlled by two PTS-catalyzed phosphorylations with antagonistic effects on
their activity. One is catalyzed by P~His-HPr, the other by one of the two
mannitol-specific PTS components (Figure 4). In addition, in certain cases
interaction of MtlR with the unphosphorylated EIIBMtl domain is also necessary to

render MtlR active [46, 47].
Figure 4. The different regulatory mechanisms controlling the activity of MtlR from B.
subtilis and G. stearothermophilus. Both proteins need to be phosphorylated by PEP, EI
and HPr at the first conserved histidine in PRD2. This phosphorylation is prevented by the
uptake of an efficiently metabolizable carbon source, such as glucose, and it serves as
CCR mechanism. MtlR from G. stearothermophilus becomes also phosphorylated by PEP,
EI, HPr, EIIAMtl and EIIBMtl at His-598 in the EIIAMtl-like domain and this
phosphorylation inhibits the transcription activation function. It is prevented when D-
mannitol is taken up via the PTS, because under these conditions the phosphoryl group of
the P~EIIBMtl domain is mainly transferred to D-mannitol bound to EIICMtl. It therefore
functions as an induction mechanism for the mtl operon. The inhibitory phosphorylation of
MtlR from B. subtilis occurs at Cys-419 in the EIIBGat-like domain and it is catalyzed by
PEP, EI, HPr and EIIAMtl. In addition, a third condition needs to be fulfilled in order to
render MtlR from B. subtilis active: It needs to be sequestered to the membrane by
interacting with the unphosphorylated EIIBMtl domain of the mannitol-specific permease
MtlA.
The first PRD-containing MtlR protein intensively studied was MtlR from
Geobacillus stearothermophilus (previously called Bacillus stearothermophilus).
The PRD-containing MtlR binds to an about 50 bp DNA region located upstream
from the mtlARFD promoter. The affinity of MtlR for its DNA target site was
strongly affected by its phosphorylation state. Phosphorylation of MtlR by PEP,
EI and HPr at the histidines in PRD2 enhanced the binding affinity about a 100-
fold compared to unphosphorylated MtlR. In contrast, when MtlR was
phosphorylated in the presence of PEP, EI, HPr, EIIAMtl and the soluble EIIBMtl
domain of MtlA the transcription activator exhibited a 10-fold lower affinity for
its operator site than unphosphorylated MtlR [44]. Under the latter conditions,
MtlR is expected to be phosphorylated at the conserved histidine(s) in PRD2 by
P~His-HPr as well as at His-598 in the EIIAMtl-like domain by P~EIIBMtl.
MtlR from G. stearothermophilus and B. subtilis exhibit 41% amino acid
sequence identity. It was therefore not surprising that both proteins become
activated by phosphorylation in PRD2 catalyzed by P~His-HPr (Figrue 4).
However, significant differences in the regulation of their activities by the
mannitol-specific PTS components were observed. Phosphorylation of the B.
subtilis PRD-containing MtlR at His-599 in the EIIAMtl-like domain by P~EIIBMtl
has only a slight inhibitory effect on its transcription activator function and a third
phosphorylation turned out to be more important. B. subtilis MtlR was the first
protein for which phosphorylation by an EIIA protein at the conserved cysteine
residue (Cys-419) in the EIIBGat-like domain has been demonstrated. This
phosphorylation exerts the main inhibitory effect on transcription activation by B.
subtilis MtlR [27]. Replacement of Cys-419 with an alanine or deletion of the
EIIAMtl-encoding mtlF gene leads to strong constitutive expression of the lacZ
reporter gene fused to the mtlA promoter [27, 48]. Surprisingly, when in addition
to mtlF the DNA fragment encoding the EIIBMtl domain of MtlA was also deleted,
MtlR was only poorly active. The EIIBMtl domain therefore seems to be required
for MtlR activity. Indeed, the unphosphorylated EIIBMtl domain of MtlA was
found to interact with the two C-terminal EIIBGat- and EIIAMtl-like domains of
MtlR and this interaction is necessary to render MtlR active [47]. Phosphorylated
EIIBMtl does not interact with MtlR [46]. However, it does not seem to be the
interaction with EIIBMtl itself that is necessary for MtlR activation, but rather the
contact with the hydrophobic membrane or a specific membrane component,
because production of EIIBMtl as a distinct soluble cytoplasmic protein inhibited
the MtlR transcription activator function.
Interestingly, EIIBMtl of V. cholerae has recently been shown to be involved
in the regulation of biofilm formation [49]. Growth on mannitol induces the
expression of the vps biofilm matrix exopolysaccharide synthesis genes.
Expression of the vps genes and biofim accumulation were also induced by
ectopic expression of the DNA fragment encoding soluble cytoplasmic EIIBMtl. It
was proposed that this mechanism allows the marine bacterium V. cholerae
attachment to mannitol producing algae and therefore would play an important
role in habitat selection.
When an efficiently metabolized PTS substrate, such as glucose, is taken up
by firmicutes, HPr will be mainly present as unphosphorylated and seryl-
phosphorylated protein and the very small amount of P~His-HPr [50] will lead to
only poor phosphorylation of MtlR in PRD2; the transcription activator will
consequently be inactive (Figure 4). This regulatory concept is supported by the
observation that B. subtilis ptsH or ptsI mutants do not express the PmtlA-lacZ
fusion [27]. The almost complete absence of P~His-HPr-mediated MtlR
phosphorylation during the uptake of glucose therefore serves as a CCR
mechanism. In contrast, the mannitol-specific EIIA and EIIB components are only
dephosphorylated when D-mannitol is transported by the PTS. The presence of D-
mannitol therefore prevents the inactivation of MtlR by phosphorylation of its
EIIBGat-like domain by P~EIIAMtl (B. subtilis) or of its EIIAMtl-like domain by the
P~EIIBMtl domain of MtlA (G. stearothermophilus) (Figure 4). The almost
complete absence of these phosphorylations in the presence of D-mannitol is
therefore used as an induction mechanism. In addition, MtlR of B. subtilis needs
to interact with the unphosphorylated EIIBMtl domain of MtlA and to be
sequestered to the membrane in order to be active (Figure 4). As explained above,
unphosphorylated EIIBMtl prevails in firmicutes when they transport D-mannitol
via the PTS. EIIBMtl-mediated MtlR sequestration to the membrane therefore
serves as a second induction mechanism.
L. casei has also been shown to transport mannitol via a PTS [51] and its
MtlR exhibits about 44% sequence similarity when compared to MtlR from G.
stearothermophilus or B. subtilis. However, in the L. casei protein the second
conserved His in PRD2 (His-399) is replaced with a tyrosine. Replacement of
either one of the two conserved histidines in PRD2 with another amino acid was
found to prevent the phosphorylation of MtlR from G. stearothermophilus and B.
subtilis by P~His-HPr [26, 27]. Similarly, preliminary experiments suggest that
owing to the His-Tyr replacement L. casei MtlR is also not phosphorylated by
P~His-HPr. In fact, in contrast to B. subtilis MtlR the L. casei regulator does not
seem to require activation via phosphorylation by PEP, EI and HPr, because
deletion of ptsI (EI) was found to lead to strong constitutive expression of the
mtlARFD operon of the lactic acid bacterium (P. Joyet and J. Deutscher,
unpublished results). In conclusion, these results demonstrate that although the
PRD-containing transcription activators of firmicutes exhibit a high degree of
sequence similarity the detailed mechanisms regulating their activity can largely
vary and need to be studied for each regulator and each species.
Owing to the absence of P~His-HPr-catalyzed phosphorylation of MtlR, CCR
of the L. casei mtlARFD operon is probably only mediated by the main catabolite
repression mechanism operative in firmicutes; it involves the catabolite control
protein A (CcpA) [52] and seryl-phosphorylated HPr (P-Ser-HPr) [53]. The
uptake of glucose leads to an increase of FBP and a decrease of inorganic
phosphate. These conditions stimulate the kinase function of the bifunctional HPr
kinase/phosphorylase (HprK/P) [54, 55] and a major part of HPr is therefore
converted into P-Ser-HPr [50]. In the presence of glycolytic intermediates P-Ser-
HPr binds to CcpA [56] and allows the transcription regulator to interact with
catabolite response elements (cre) [57]. These operator sites precede most
catabolic transcription units and binding of CcpA/P-Ser-HPr either inhibits or
stimulates their expression leading to CCR or carbon catabolite activation (CCA),
respectively. The mtl operon of B. subtilis has indeed been shown to be submitted
to CcpA/P-Ser-HPr-mediated CCR. Mutants deleted for hprK, the gene encoding
HprK/P, or producing a mutant HPr in which Ser-46 is replaced with an alanine
exhibit strongly reduced CCR for the mtl operon [50, 58]. In B. subtilis, the
remaining repression is due to the above described MtlR-mediated CCR. CCR of
the mtl operon in enterobacteriaceae is probably mediated by the complex formed
by cyclic-AMP and the cAMP receptor protein (cAM/Crp), which stimulates the
expression of catabolic genes. In enterobacteriaceae, the uptake of an efficiently
utilized carbon source, such as glucose, lowers the cAMP level [59] and the
cAMP/Crp complex is no longer formed. As a consequence catabolic genes are
only poorly expressed. Competition of the glucose- and mannitol-specific PTSs
for the common phosphoryl donor P~His-HPr has also been discussed as a CCR
mechanism for carbohydrates taken up via a PTS. According to this concept,
P~His-HPr would donate its phosphoryl group preferentially to EIIAGlc and less
frequently to other EIIAs, such as EIIAMtl.
Although several actinobacteria transport D-mannitol via a PTS (MtlA) and
many of them possess PRD-containing transcription activators, in none of them is
the expression of the mannitol operon controlled by a PRD-containing MtlR. The
mtlAD PTS operon of several Arthrobacter and Leifsoniae species is preceded by
a gene encoding a repressor of the TetR family, which probably controls the
expression of the mannitol operon. No gene encoding a putative transcription
regulator is found in the vicinity of the D-mannitol PTS operon in several other
actinobacteria and the regulation of their expression remains obscure. As already
mentioned before, the mtlTD operon present in corynebacteria is usually
controlled by a DeoR-like repressor.
PRODUCTION OF D-MANNITOL BY BACTERIA

D-Mannitol is widely used as low-calory sweetener in all kinds of food
products [60] and in medicine as a potent osmotic diuretic [61]. Owing to its
noncariogenic properties it is also used in breath-freshning products and sugar-
free chewing gums. D-Mannitol is presently mainly produced by the chemical

hydrogenation of fructose to mannitol at 130 to 150C and high pressure with
hydrogen gas and Ni2+ as catalyst. The increasing demand for D-mannitol initiated
a search for more specific and less expensive procedures including its
biotechnological production by bacteria.
As already mentioned, several bacteria, mainly heterofermentative lactic acid
bacteria of the genera Lactobacillus, Leuconostoc and Oenococcus, are able to
naturally produce D-mannitol [3, 13, 62]. Under specific growth conditions these
organisms can use fructose as an alternative electron receptor. For this purpose
they possess the enzyme D-mannitol 2-dehydrogenase (Mdh) that catalyzes the
NADPH-dependent reduction of fructose to D-mannitol. The Mdh protein of
several organisms has been purified and characterized [63-65]. The Zn2+-
dependent enzyme has a MW of about 40 kDa, but despite the similar size it
exhibits no sequence similarity to mannitol-1-P 5-dehydrogenases, MtlD(s), of
firmicutes or the larger mannitol 2-dehydrogenases MtlD(l) from corynebacteria
or pseudomonodaceae [64].
The affinity of the enzyme for NADH is significantly lower than for NADPH.
For the industrial production of mannitol by Mdh-containing bacteria the
lactobacilli [66] and Leuconostoc strains are usually grown on cheap carbon
sources, such as carob syrup [67] or sugarcane molasses [68, 69], alone or
combined with fructose syrup. By using Lactobacillus reuteri as organism and
high concentrations of sugarcane molasses the D-mannitol concentration in the
supernatant reached more than 200 mM with an about 90% yield [68]. Nothing is
known how the intracellularly produced D-mannitol is secreted by these bacteria
into the medium.
Interestingly, in several heterofermentative lactic acid bacteria three genes
located downstream from mdh encode an ABC efflux system [63], which might
possibly function as D-mannitol exporter. Attempts were also made to use
purified Mdh for biocatalytic hydrogenation of D-fructose to D-mannitol. A
problem was the regeneration of the cofactor NAD(P)H, which was tried to be
solved by the concomitant NADH-producing oxidation of glucose to gluconate
[70].
In contrast to heterofermentative lactic acid bacteria homofermentative
organisms are not able to produce significant amounts of D-mannitol. It was
therefore attempted to genetically modify certain homofermentative lactic acid
bacteria, such as Lactococcus lactis, in order to make them produce D-mannitol.
In fact, only mutants with an impaired ability to regenerate NAD+ owing for
example to a deletion of the lactate dehydrogenase (ldh) gene are able to produce
D-mannitol. L. lactis double mutants defective in ldh and in mtlA (EIICBMtl) or
mtlF (EIIAMtl) were constructed and were indeed found to produce intracellular
D-mannitol from glucose at a concentration of about 70 mM and a yield of 30% to
40% [71, 72]. In another attempt, D-mannitol-1-P dehydrogenase and D-mannitol
1-phosphatase were produced in an ldh deletion strain of L. lactis. This allowed
the transformation of D-fructose-6-P into D-mannitol-1-P followed by its
dephosphorylation to D-mannitol with an about 50% yield [73]. Other organisms,
such as E. coli, were also genetically modified for improved D-mannitol
production. Usually the mannitol-2 dehydrogenase, Mdh, from different microbes
was synthesized in the genetically modifed strains [3, 17].
Finally, it should be mentioned that bacterial D-mannitol-1-P 5-
dehydrogenases have been introduced into transgenic plants, such as tobacco [74],
in order to transform D-fructose-6-P into D-mannitol-1-P, which is subsequently
dephosphorylated by the plant D-mannitol 1-phosphatase to mannitol. The
increased production of D-mannitol by the plants provided a protection against
osmolytic stress and droughts.
CONCLUSION
D-Mannitol, the most abundant sugar alcohol in nature, is an important
carbon and energy source for bacteria. This is underlined by the fact that during
evolution bacteria developped at least three different types of D-mannitol-specific
transport systems, two different types of catabolic routes and four different modes
of regulation of mtl operon expression. Especially the uptake and phosphorylation
of the hexitol by the PTS allows its efficient utilization. Nevertheless, glucose and
probably a few other carbohydrates are preferentially utilized and repress the
synthesis of the enzymes necessary for D-mannitol metabolism.
A few bacteria, mainly heterofermentative lactic acid bacteria, are able to
produce themselves D-mannitol from D-fructose. Seen the increasing use of D-
mannitol in the food industry and in medicine, the biotechnological production of
the hexitol by genetically modified bacteria growing on cheap carbon sources,
such as sugar cane melasses, is expected to significantly lower the costs of its
production. However, the organisms genetically modified so far still need to be
further optimized in order to make the biotechnological synthesis competitive
with the present chemical production of D-mannitol by reduction of fructose. The
synthesis of D-mannitol in plants normally not producing this hexitol and its use
as osmoprotectant and as protectant during droughts is very promising as well, but
also needs further optimization.
REFERENCES
[1] J. Wang, Y. M. Kim, H. S. Rhee, M. W. Lee and J. M. Park, Bioresour.
Technol. 135, 199 (2013).
[2] J. M. H. Stoop, J. D. Williamson and D. M. Pharr, Trends in Plant Science
1, 139 (1996).
[3] S. M. Bhatt, A. Mohan and S. K. Srivastava, ISRN Biotechnology Article ID
914187, dx.doi.org/10.5402/2013/914187 (2013).
[4] S. Rousvoal, A. Groisillier, S. M. Dittami, G. Michel, C. Boyen and T.
Tonon, Planta 233, 261 (2011).
[5] R. H. Reed and P. J. Wright, Mar. Ecol. Prog. Ser. 29, 205 (1986).
[6] K. Iwamoto and Y. Shiraiwa, Mar. Biotechnol. (NY) 7, 407 (2005).
[7] E. Oddo, F. Saiano, G. Alonzo and E. Bellini, Ann. Bot. (Lond.) 90, 239
(2002).
[8] W. H. Loescher, R. H. Tyson, J. D. Everard, R. J. Redgwell and R. L.
Bieleski, Plant Physiol. 98, 1396 (1992).
[9] P. Delavault, P. Simier, S. Thoiron, C. Vronsi, A. Fer and P. Thalouarn,
Physiol. Plant 115, 48 (2002).
[10] H. Vlz, N. J. Glassbrook and M. E. Daub, Fungal Genet. Biol. 44, 258
(2007).
[11] J. M. Stoop and D. M. Pharr, Arch. Biochem. Biophys. 298, 612 (1992).
[12] W. J. Wannet, R. W. Wassenaar, H. J. Jorissen, C. van der Drift and H. J.
Op den Camp, Antonie Van Leeuwenhoek 77, 215 (2000).
[13] M. E. Ortiz, J. Bleckwedel, R. R. Raya and F. Mozzi, Appl. Microbiol.
Biotechnol. 97, 4713 (2013).
[14] X. Peng, N. Okai, A. A. Verts, K. Inatomi, M. Inui and H. Yukawa, Appl.
Microbiol. Biotechnol. 91, 1375 (2011).
[15] L. Ribas De Pouplana, S. Atrian, R. Gonzlex-Duarte, L. A. Fothergill-
Gilmore, S. M. Kelly and N. C. Price, Biochem. J. 276, 433 (1991).
[16] P. Brnker, J. Altenbuchner and R. Mattes, Gene 206, 117 (1998).
[17] P. Brnker, J. Altenbuchner, K. D. Kulbe and R. Mattes, Biochim. Biophys.
Acta 1351, 157 (1997).
[18] J. Deutscher, C. Francke and P. W. Postma, Microbiol. Mol. Biol. Rev. 70,
939 (2006).
[19] B. Reiche, R. Frank, J. Deutscher, N. Meyer and W. Hengstenberg,
Biochemistry 27, 6512 (1988).
[20] J. Reizer, S. L. Sutrina, L.-F. Wu, J. Deutscher, P. Reddy and M. H. Saier,
Jr., J. Biol. Chem. 267, 9158 (1992).
[21] A. L. Honeyman and R. Curtiss III, Microbiology 146, 1565 (2000).
[22] A. Maz, G. Bol, M. Ziga, A. Bourand, V. Loux, M. J. Yebra, V.

Monedero, K. Correia, N. Jacques, S. Beaufils, S. Poncet, P. Joyet, E.
Milohanic, S. Casargola, Y. Auffray, G. Prez-Martnez, J. F. Gibrat, M.
Zagorec, C. Francke, A. Hartke and J. Deutscher, J. Bacteriol. 192, 2647
(2010).
[23] R. Fischer, R. Pogge von Strandmann and W. Hengstenberg, J. Bacteriol.
173, 3709 (1991).
[24] F. M. Ak, P. Joyet, J. Deutscher and E. Milohanic, Mol. Microbiol. 81, 274
(2011).
[25] R. Pogge von Strandmann, C. Weigt, R. Fischer, H. E. Meyer, H. R.
Kalbitzer and W. Hengstenberg, Eur. J. Biochem. 233, 116 (1995).
[26] S. A. Henstra, R. H. Duurkens and G. T. Robillard, J. Biol. Chem. 275,
7037 (2000).
[27] P. Joyet, M. Derkaoui, S. Poncet and J. Deutscher, Mol. Microbiol. 76, 1279
(2010).
[28] H. H. Pas and G. T. Robillard, Biochemistry 27, 5835 (1988).
[29] J. Y. Suh, M. Cai, D. C. Williams Jr and G. M. Clore, J. Biol. Chem. 281,
8939 (2006).
[30] G. Cornilescu, B. R. Lee, C. C. Cornilescu, G. Wang, A. Peterkofsky and G.
M. Clore, J. Biol. Chem. 277, 42289 (2002).
[31] G. R. Jacobson, C. A. Lee and M. H. Saier Jr, J. Biol. Chem. 254, 249
(1979).
[32] E. P. Vos, R. ter Horst, B. Poolman and J. Broos, Biochim. Biophys. Acta.
1788, 581 (2009).
[33] B. A. van Montfort, S.-W. G.K., J. Wind, J. Broos, G. T. Robillard and B.
Poolman, J. Biol. Chem. 277, 14717 (2002).
[34] C. A. Lee and M. H. Saier Jr, J. Biol. Chem. 258, 10761 (1983).
[35] M. Opaci, E. P. Vos, B. H. Hesp and J. Broos, J. Biol. Chem. 285, 25324
(2010).
[36] S. Watanabe, M. Hamano, H. Kakeshita, K. Bunai, S. Tojo, H. Yamaguchi,
Y. Fujita, S. L. Wong and K. Yamane, J. Bacteriol. 185, 4816 (2003).
[37] S. Otte and J. W. Lengeler, FEMS Microbiol. Lett. 194, 221 (2001).
[38] P. Rambhatla, S. Kumar, J. T. Floyd and M. F. Varela, J. Microbiol.
Biotechnol. 21, 914 (2011).
[39] R. M. Figge, T. M. Ramseier and M. H. Saier, Jr., J. Bacteriol. 176, 840
(1994).
[40] K. Tan, S. Clancy, M. Borovilos, M. Zhou, S. Hrer, S. Moy, L. L. Volkart,
J. Sassoon, U. Baumann and A. Joachimiak, J. Biol. Chem. 284, 36670
(2009).
[41] L. M. Mustachio, S. Aksit, R. H. Mistry, R. Scheffler, A. Yamada and J. M.

Liu, J. Bacteriol. 194, 598 (2012).
[42] P. Brnker, M. Hils, J. Altenbuchner and R. Mattes, Gene 215, 19 (1998).
[43] M. T. Gallegos, R. Schleif, A. Bairoch, K. Hofmann and J. L. Ramos,
Microbiol. Mol. Biol. Rev. 61, 393 (1997).
[44] S. A. Henstra, M. Tuinhof, R. H. Duurkens and G. T. Robillard, J. Biol.
Chem. 274, 4754 (1999).
[45] S. Behrens, W. Mitchell and H. Bahl, Microbiology 147, 75 (2001).
[46] P. Joyet, H. Bouraoui, F. M. Ak, M. Derkaoui, A. C. Zbr, T. N. Cao, M.
Ventroux, S. Nessler, M. F. Noirot-Gros, J. Deutscher and E. Milohanic,
Biochim. Biophys. Acta 1834, 1415 (2013).
[47] H. Bouraoui, M. Ventroux, M.-F. Noirot-Gros, J. Deutscher and P. Joyet,
Mol. Microbiol. 87, 789 (2013).
[48] K. M. Heravi, M. Wenzel and J. Altenbuchner, Microb. Cell Fact. 10, 83
(2011).
[49] P. Ymele-Leki, L. Houot and P. I. Watnick, Appl. Environ. Microbiol. 79,
[Epub ahead of print] (2013).
[50] V. Monedero, S. Poncet, I. Mijakovic, S. Fieulaine, V. Dossonnet, I.
Martin-Verstraete, S. Nessler and J. Deutscher, EMBO J. 20, 3928 (2001).
[51] R. Viana, V. Monedero, V. Dossonnet, C. Vadeboncoeur, G. Prez-
Martnez and J. Deutscher, Mol. Microbiol. 36, 570 (2000).
[52] T. M. Henkin, F. J. Grundy, W. L. Nicholson and G. H. Chambliss, Mol.
Microbiol. 5, 575 (1991).
[53] J. Deutscher, Curr. Opin. Microbiol. 11, 87 (2008).
[54] J. Deutscher and M. H. Saier, Jr., Proc. Natl. Acad. Sci. USA 80, 6790
(1983).
[55] I. Mijakovic, S. Poncet, A. Galinier, V. Monedero, S. Fieulaine, J. Janin, S.
Nessler, J. A. Marquez, K. Scheffzek, S. Hasenbein, W. Hengstenberg and
J. Deutscher, Proc. Natl. Acad. Sci. USA 99, 13442 (2002).
[56] J. Deutscher, E. Kster, U. Bergstedt, V. Charrier and W. Hillen, Mol.
Microbiol. 15, 1049 (1995).
[57] Y. Fujita, Y. Miwa, A. Galinier and J. Deutscher, Mol. Microbiol. 17, 953
(1995).
[58] J. Deutscher, J. Reizer, C. Fischer, A. Galinier, M. H. Saier, Jr. and M.
Steinmetz, J. Bacteriol. 176, 3336 (1994).
[59] R. S. Makman and E. W. Sutherland, J. Biol. Chem. 240, 1309 (1965).
[60] V. Monedero, G. Prez-Martnez and M. J. Yebra, Appl. Microbiol.
Biotechnol. 86, 1003 (2010).
[61] B. C. Saha and F. M. Racine, Appl. Microbiol. Biotechnol. 89, 879 (2011).
[62] B. C. Saha and F. M. Racine, Appl. Microbiol. Biotechnol. 87, 553 (2010).
[63] Y. Sasaki, M. Laivenieks and J. G. Zeikus, Appl. Microbiol. Biotechnol. 68,
36 (2005).
[64] J. Aarnikunnas, K. Rnnholm and A. Palva, Appl. Microbiol. Biotechnol.
59, 665 (2002).
[65] G. Hahn, B. Kaup, S. Bringer-Meyer and H. Sahm, Arch. Microbiol. 179,
101 (2003).
[66] C. Rodrguez, T. Rimaux, M. J. Fornaguera, G. Vrancken, G. F. de Valdez,
L. De Vuyst and F. Mozzi, Appl. Microbiol. Biotechnol. 93, 2519 (2012).
[67] F. Carvalheiro, P. Moniz, L. C. Duarte, M. P. Esteves and F. M. Grio, J.
Ind. Microbiol. Biotechnol. 38, 221 (2011).
[68] M. E. Ortiz, M. J. Fornaguera, R. R. Raya and F. Mozzi, Appl. Microbiol.
Biotechnol. 95, 991 (2012).
[69] B. C. Saha, Appl. Microbiol. Biotechnol. 72, 676 (2006).
[70] M. Howaldt, A. Gottlob, K. D. Kulbe and H. Chmiel, Ann. N.Y. Acad. Sci.
542, 400 (1989).
[71] P. Gaspar, A. R. Neves, M. J. Gasson, C. A. Shearman and H. Santos, Appl.
Environ. Microbiol. 77, 6826 (2011).
[72] P. Gaspar, A. R. Neves, A. Ramos, M. J. Gasson, C. A. Shearman and H.
Santos, Appl. Environ. Microbiol. 70, 1466 (2004).
[73] H. W. Wisselink, A. P. Moers, A. E. Mars, M. H. Hoefnagel, W. M. de Vos
and J. Hugenholtz, Appl. Environ. Microbiol. 71, 1507 (2005).
[74] M. C. Tarczynski, R. G. Jensen and H. J. Bohnert, Science 259, 508 (1993).
In: Mannitol ISBN: 978-1-62808-762-8
Chapter 2
CONCENTRATION OF MANNITOL
AND OTHER SOLUBLE CARBOHYDRATES
IN THE CRUSTOSE LICHEN RHIZOCARPON
GEOGRAPHICUM
Richard A. Armstrong
Department of Vision Sciences, Aston University, Birmingham, UK
ABSTRACT
In symbiotic lichens which have Trebouxia as the algal partner,
photosynthesis by the algae results in the production of the soluble
carbohydrate ribitol which is then transported to the fungus where it is
converted to arabitol and mannitol. Within the fungus, arabitol may act as a
short-term carbohydrate reserve while mannitol may have a more protective
function and be important in stress resistance. The concentrations of ribitol,
arabitol, and mannitol were measured, using gas chromatography, in the
central areolae and marginal hypothallus of the crustose lichen Rhizocarpon
geographicum (L.) DC. growing on slate rocks in north Wales, UK. The
concentrations of all three soluble carbohydrates were greater in the central
areolae than in the marginal prothallus. In addition, the ratio of mannitol in
the prothallus to that in the areolae was least in July. The concentration of an
individual carbohydrate in the prothallus was correlated primarily with the
Corresponding author: R.A. Armstrong Tel. +44-121-204-4102, Fax. +44-121-204-4048, Email.

R.A.Armstrong@aston.ac.uk.
22 Richard A. Armstrong
concentrations of the other carbohydrates in the prothallus and not to their

concentrations in the areolae. Low concentration of ribitol, arabitol, and
mannitol in the marginal prothallus compared with the central areolae
suggests either a lower demand for carbohydrate by the prothallus or limited
transport from areolae to prothallus and may explain the low growth rates of
this species. In addition, soluble carbohydrates appear to be partitioned
differently through the year with an increase in mannitol compared with
arabitol in more stressful periods.
Keywords: Lichen, symbiosis, Rhizocarpon geographicum, mannitol, prothallus,

areolae, soluble carbohydrate
INTRODUCTION
A lichen is an intimate association between an alga and a fungus and regarded
as one of the best examples of mutualism or symbiosis involving
microorganisms (Armstrong, 2011). The lichen thallus is highly structured but
in different species shows varying degrees of integration of the two symbionts. A
typical lichen is composed mainly of fungal hyphae with eucaryotic algal cells
embedded in an upper cortical layer. The algal partner carries out photosynthesis
and supplies the fungus with carbohydrate but there is little experimental evidence
to suggest that the fungus supplies nutrients directly to the alga (Smith and
Douglas, 1987). If there is a benefit to the alga, it may lie in the protection offered
by the thallus, thus extending the range of habitats that can be potentially
occupied by the alga.
There are three common lichen growth forms. In fruticose lichens, the thallus
is attached to the substratum at a single point and forms a complex branched
structure. By contrast, in foliose lichens, the thallus comprises a series of radially
arranged leaf-like marginal lobes, while crustose-type lichens comprise a thin
crust tightly attached to the surface of rock or tree bark. Endolithic lichens, in
which the lichen lives within the surface layers of the rock, are the most extreme
example of the crustose lifestyle (Armstrong and Bradwell, 2010). In endolithic
species, the upper cortex is absent while the algae and fungal hyphae are scattered
within the surface layers of the substratum. Most crustose species, however, have
a distinct upper cortex, an algal layer, and fungal medulla. In some species, the
margin of the thallus is diffuse and not sharply demarcated, but in others is
delimited by a non-lichenized fungal prothallus. One of the most widely
distributed species of the latter type is Rhizocarpon geographicum (L.) DC.
Mannitol in Rhizocarpon geographicum 23
(Figure 1) (Armstrong, 2011). This unusual organism comprises yellow-green

areolae growing in association with a non-lichenized fungal prothallus that
extends beyond the margin of the areolae to form a peripheral ring. This species
grows exceptionally slowly and its considerable longevity has been exploited by
geologists in the development of methods of dating the age of exposure of rock
surfaces and glacial moraines (lichenometry) (Armstrong and Bradwell, 2010).
R. geographicum may represent one of the most primitive types of growth form in
lichens (Armstrong, 2011).
STRUCTURE OF RHIZOCARPON
R. geographicum consists of a flat basal plate of black fungal tissue termed
the prothallus. Discreet areolae (Figure 2), containing the algal cells, develop in
association with this fungal prothallus but the prothallus extends beyond the outer
edge of the areolae to form a marginal ring normally 1 - 3 mm in width
(Armstrong and Bradwell, 2001).
Figure 1. Thalli of Rhizocarpon geographicum (L.) DC. growing on a slate rock surface in
north Wales, UK. The non-lichenized black prothallus is clearly visible especially at the
margin of the thalli (Bar = 1 cm).
Figure 2. Structure of a typical areole of the lichen Rhizocarpon geographicum (L.) DC.
Note the colourless cuticle covering the cortex the algae occur in clusters in a distinct layer
below the cortex. Throughout the areolae, fungal hyphae grow vertically upwards from the
basal region.
The areolae are highly variable in shape and morphological differences

between them may be attributable to their origin. New areolae developing on the
fungal prothallus are generally punctate or verrucose (warty) in shape while
mature areolae in the centre of the thallus are often lobed in shape. Within each
areola, there is a superficial transparent cuticle, a cortical layer of fungal hyphae
10-80 m in depth, an algal layer consisting of clusters of single cells of the green
alga Trebouxia, and fungal medullary tissue. The organism grows on the rock
substratum by radial extension of the prothallus.
Development of Rhizocarpon
Rhizocarpon geographicum poses several interesting biological problems.

First, how is the primary thallus formed on a substratum? Second, once formed,
how does the prothallus develop areolae both at the margin and in the centre of
the thallus and third, how is carbohydrate supplied to the marginal prothallus?
Four processes contribute to the development of a mature thallus of R.

geographicum, viz., the formation of the primary areolae, growth and division of
areolae, the confluence of areolae, and the fusion of individual thalli to form
larger individuals (Asta & Letrouit-Galinou, 1995). The development of primary
areolae and the formation of the prothallus in a related species, viz., Rhizocarpon
lecanorinum, have been studied by Clayden (1998). The first identifiable stage in
development is a compact granule in which fungal hyphae have become
associated with a compatible species of Trebouxia. Thallus differentiation occurs
resulting in the formation of a typical areole. The process of differentiation is
associated with the formation and deposition of rhizocarpic acid, a secondary
lichen substance found in the incipient cortical layer in the apical part of the
granule.
The radiating prothallus is initiated from the basal margin of the primary
areole, growing out to form a marginal ring. Removal of the marginal prothallus
in a mature R geographicum thallus by scraping away the fungal tissue with a
scalpel, however, results in regeneration of the prothallus but by a different
mechanism. The new prothallus is formed first, by retreat of the outer margin of
the areolae and second, by new hyphal growth (Armstrong and Smith, 1987).
Hence, the presence of a marginal prothallus appears to be so important that
areolae at the edge appear to be sacrificed to enable the prothallus to survive.
After the primary areolae and marginal prothallus are formed, new areolae
continue to develop, most frequently on the marginal prothallus, as it advances.
A number of processes may be involved in the formation of these new areolae.
First, Nienberg (1926) observed in the crustose lichen genus Pertusaria that algal
cells originating in the areolae were pushed into the growing area. Hence, the
thallus a few millimetres from the edge was composed of radially elongated
hyphae and a few migrating algal cells were pushed forwards by specialised
hyphae. Second, Slocum et al. (1980) observed that the alga Trebouxia could form
zoospores within the lichen thallus that could swim from the central areolae to
colonize the prothallus. Third, zoospores from neighbouring thalli could swim to
the prothallus and initiate the areolae and fourth, the prothallus could trap free-
living algal cells on the substratum as it advanced. The development of new
areolae on the marginal prothallus was studied experimentally by Armstrong &
Smith (1987). New areolae were slow to develop on the marginal prothallus but
formed at a similar rate whether or not the central areolae were completely
removed or separated from the marginal prothallus by a 2 mm or 5 mm wide
moat. There was no evidence, therefore, that the central areolae were involved in
the formation of the new areolae on the marginal prothallus. It was concluded that
areolae at the margin may develop from free-living algal cells trapped by the
prothallus. Free-living Trebouxia cells are often the first to colonize a bare
substratum and such cells can be detected on the surface before any lichen thallus
has become established (Mukhtar et al., 1994).
Growth of Rhizocarpon
Rhizocarpon geographicum is one of the slowest growing of all crustose

lichens (Table 1) and in arctic and alpine locations, radial growth rates (RaGR) of
less than 0.1 mm yr-1 have been recorded (Armstrong, 2005). To explain slow
growth, it is necessary to establish how the marginal prothallus obtains its
carbohydrate supply for growth. Materials for growth could be obtained from the
central areolae either by translocation through fungal hyphae or by leakage and
reabsorption, from pioneer algal cells trapped within the prothallus, or from
exogenous sources such as surface runoff.
Table 1. Examples of the reported growth rates (radial growth rate,

RGR, mm yr-1) for thalli of the lichen Rhizocarpon geographicum
from various habitats
Location RGR (mm yr-1) Author

South Orkney, Ant 0.1 Hooker, 1980
West Greenland 0.05 0.1 Beschel, 1958
Alaska 0 0.18 Haworth, 1986
Washington State, USA 0 0.19 Armstrong, 2005
BC, Canada 0.26 0.41 McCarthy, 2003
New Hampshire, USA 0.4 Hausmann, 1948
Maritime Antarctic 0.50 Sancho, 2004
Switzerland 0.50 maximum Proctor, 1983
North Labrador, Canada 0.10 0.58 Rogerson, 1986
South Norway 0.66 Mathews, 1994
North Wales, UK 0.74 Winchester &
Chaujar, 2002
North Wales, UK 0.03 - 0.94 Armstrong, 1983
Field experiments have suggested that the prothallus of R. geographicum may

have the ability to utilize exogenous nutrients (Armstrong and Smith, 1996). On a
south-facing rock surface in north Wales, for example, the areolae were scrapped
away to isolate the peripheral prothallus. Hypothalli without any observable
areolae grew at similar rates as adjacent, intact thalli for a period of two months,
but growth then declined and the hypothalli fragmented and disappeared from the
surface within six months. Hence, if the prothallus is using exogenous supplies of
carbohydrate, concentrations on the surface appear to be insufficient to maintain
growth other than for short periods of time.
An alternative explanation for the results, however, is that the prothallus is
using carbohydrate reserves in the periphery for growth and once these were
exhausted, the fungal hyphae die. In a further experiment (Armstrong and Smith,
1987), individual thalli of R. geographicum were removed from rock surfaces,
each on a small piece of smooth slate. Complete removal of the central areolae
resulted in no measurable RaGR of the prothallus over a period of 18 months.
Removal of the areolae to within 1 and 2 mm of the prothallus, however,
significantly reduced growth in proportion to the width of the areolae present.
These results suggest that carbohydrate is supplied to the marginal prothallus by
the central areolae and that pioneer algal cells trapped in the prothallus do not
produce sufficient carbohydrate for growth processes.
Hence, the slow growth of R. geographicum could be a consequence of its
primitive growth form and a direct result of the problem of transferring
carbohydrate from the central areolae to the marginal prothallus. Nutrient transfer
may occur only within the immediate vicinity of each individual areola (Innes,
1985) and therefore mature areolae located at the margin may be the most
important contributor to the growth of the prothallus.
Carbohydrates in Rhizocarpon
Soluble carbohydrate may account for up to 10% of thallus dry weight in

some lichens (Lewis and Smith, 1967; Dudley and Lechowitz, 1987; Macfarlane
and Kershaw, 1985). In Trebouxia containing lichens, such as R. geographicum,
carbohydrate is released from the alga as the soluble polyol ribitol, and is then
converted into arabitol and mannitol by the fungus (Richardson et al., 1968).
These carbohydrates may have several roles in lichens including carbohydrate
metabolism, contributing to the polymeric reserve, and in providing metabolic
protection for membranes and proteins under conditions of stress (Farrar, 1988).
In addition, where two or more carbohydrates are commonly present, each may
have a distinct role. Arabitol is depleted more rapidly than mannitol under
conditions of stress suggesting arabitol acts a short-term carbohydrate reserve
while mannitol has a more protective function (Farrar, 1983). The mechanism by
which carbohydrates are transferred from the algal cells in the areolae to the
fungal prothallus is unknown in Rhizocarpon-type lichens. In some foliose and

fruticose lichens, transfer occurs within an extra-cellular envelope of hydrophobic
proteinaceous material creating an apoplastic continuum but this mechanism is
unlikely to be present in crustose lichens (Honegger, 1998). In addition, algal cells
in R. lecanorinum are not penetrated by haustoria, a mechanism which can extract
nutrients in those lichens which have a less integrated thallus organisation than
members of the genus Rhizocarpon (Clayden, 1998).
The marginal prothallus may obtain its carbohydrate for growth: (1) from the
areolae by transport through the hyphae, (2) from pioneer algal cells embedded
in the prothallus (Nienburg, 1926), (3) by leakage from the areolae followed by
reabsorption (Farrar and Smith, 1976), or (4) from exogenous sources such as
surface run off.
Previous experiments, however, suggest that neither pioneer algae cells nor
exogenous sources of carbohydrate can support the radial growth of the marginal
prothallus (Armstrong and Smith, 1996). Hence, the marginal prothallus may
obtain its carbohydrate from the central areolae via hyphal connections
(Armstrong and Smith, 1987). There have been few studies of the concentrations
of carbohydrates in the yellow-green species of Rhizocarpon. The concentrations
of the three major soluble carbohydrates ribitol, arabitol, and mannitol were
measured in the central areolae, and marginal hypothalli in north Wales,
concentrations being several times higher in the areolae than the marginal
prothallus (Armstrong and Smith, 2009).
Objectives
A number of aspects of soluble carbohydrate metabolism may be important in

determining lichen growth and in explaining lichen distribution. Hence, a
reduction in RaGR on a particular occasion may result from a decrease in
carbohydrate synthesis by the algal partner, an increase in the allocation of
carbohydrate to arabitol and mannitol in the fungus, or a depletion of
carbohydrate under conditions of stress.
The objectives of the present study were: (1) to measure the concentrations of
the principal soluble carbohydrates present in R. geographicum, viz., ribitol,
arabitol, and mannitol, (2) to compare the concentrations of carbohydrates in the
central areolae and marginal prothallus, (3) to compare the relative concentrations
of carbohydrates between sample periods, and (4) to study the correlations
between concentrations of the ribitol, arabitol, and mannitol both within and
between prothallus and areolae.
MATERIALS AND METHODS

Study Site
The study was carried out at a site in South Gwynedd, north Wales (Grid Ref.
SN 6196) in an area of Ordovician slate rock (Armstrong, 1974). Slate outcrops
varying in surface area from 2 30 m2 are a common feature of the hillsides in
this region. These surfaces possess a rich lichen flora characteristic of siliceous
rock in the north and west of the UK (James et al., 1977) and include communities
with a high proportion of crustose species (Armstrong, 1974). R. geographicum is
a member of several different communities at the site, especially on south-facing
rock surfaces (Armstrong, 1974; 2002).
Taxonomy of Rhizocarpon
Identification to species can be difficult in the yellow-green Rhizocarpon

group especially in the subgenus Rhizocarpon Ram. em. Th. Fr. subgen.
Rhizocarpon. This subgenus is subdivided into four sections, viz., Superficiale,
Alpicola, Viridiatrum and Rhizocarpon (Poelt, 1988).
Thalli can be identified to section level fairly easily using the identification
criteria suggested by Benedict (1988). Hence, all thalli included in the study were
identified as Rhizocarpon section Rhizocarpon (L.) DC., i.e., spores were greater
than two-celled, the epihymenium was not black, and the medulla was intense
blue/violet when treated with a 1% solution of iodine (Benedict, 1988).
By contrast, identification to species level within a section is extremely
difficult, especially in the Rhizocarpon section. Using the broadly circumscribed
criteria of Purvis et al. (1992), however, the thalli included in this study were all
identified as R. geographicum (L.) DC.
Sampling of R. Geographicum
Samples of areolae and prothallus were obtained from a population of R.

geographicum thalli maintained for growth experiments at the site (Armstrong,
2002; Armstrong and Smith, 1996; Armstrong and Bradwell, 2001). These thalli
were collected originally on pieces of smooth slate from south-facing rock
surfaces and placed in full sun on flat wooden boards in the field. Thalli were
allowed to equilibrate for a year before measurement of carbohydrates was made.
No deleterious effects of the transplant procedure were observed and the thalli
grew on the boards at similar rates to those in situ on south-facing rock surfaces
(Armstrong, 2002). Samples of areolae and prothallus were collected from
randomly chosen thalli on 1 November 1997, 1 February 1998, 1 April 1998, and
1 July 1998. Four replicate samples of the marginal prothallus and areolae (10
50 mg) were then obtained on each occasion by gently scraping the thalli with a
scalpel under a dissecting microscope. Samples of prothallus were taken as close
to the growing edge as possible and of areolae within 1 cm of the marginal
prothallus. Care was taken to avoid contaminating the prothallus samples with
developing areolae. Samples were stored in 80% ethanol in a refrigerator and
were analysed within one week of collection.
Measurement of Carbohydrates
The concentrations of carbohydrates were determined by gas chromatography

following the procedures of Holligan (1971) and Holligan and Drew (1971).
Samples of prothallus and areolae (3.5 40 mg dry weight) were extracted with
three changes of 80% ethanol. The extracts were reduced to near dryness under
reduced pressure and made up to 2 ml with distilled water. Extracts were then
shaken with Amberlite IR120 (H+) and IR45 (OH-) ion exchange resins (BDH
UK) for 20 min to remove amino and organic acids (Whipps and Cooke, 1978).
Extracts were transferred to new flasks, reduced to dryness under reduced
pressure, and silylated with bis(trimethysilyl) trifluoroacetamide (BSFTA) and
trimethylchlorosilane (TMCS) in anhydrous pyridine. After 18 hours, the resulting
carbohydrate (TMS) derivatives were separated on a 25 m x 0.32 mm Flexsil
capillary column overlain with 0.4 l OV1 liquid phase (Phase Separations UK).
1 l aliquots of each sample were initially injected onto a pre-column of silanised
glass beads (100 mesh) and then split in the ratio 1:4 to avoid overloading the
capillary column.
Operating conditions of the Varian 3700 gas chromatograph were: injection
temperature 150C, oven temperature 150-270C rise/min; FID detector
temperature 320C. Characterisation and quantification of unknown derivatives
were performed by comparison and chromatography with known carbohydrate
standards. Data analysis (t tests, analysis of variance, Pearsons correlation
coefficient, stepwise multiple regression) was carried out using STATISTICA
software (Statsoft Inc., 2300 East 14th St, Tulsa, OK 74104, USA).
CONCENTRATION OF CARBOHYDRATES
IN RHIZOCARPON
A typical gas chromatogram trace obtained from a sample of areolae collected

on 1 July is shown in Figure 3. Of these peaks, those labelled 1-3 remain
unidentified, peak 4 was identified as arabitol, peak 5 as ribitol, peak 7 as
fructose, peak 8 as -glucose, while the large peak 9 was mannitol. The remaining
peaks present were identified as -glucose (peak 10), sucrose (peak 11), and
trehalose (peak 12). There was also a small peak adjacent to peak 7 which remains
unidentified.
Figure 3. A typical gas chromatogram trace obtained from a sample of areolae collected on
1 July. Peak 4 is arabitol, peak 5 ribitol, and peak 9 mannitol.
The concentrations of ribitol, arabitol, and mannitol in the prothallus and

areolae are shown in Table 2. The mean concentrations of arabitol (t = 6.31, P <
0.001), ribitol (t = 4.41, P < 0.001), mannitol (t = 4.11, P < 0.001), and -glucose
(t = 2.62, P < 0.05), averaged over sample times, were significantly greater in the
areolae than in the prothallus.
Table 2. Mean concentration (g g-1 extracted tissue, SEM in parentheses)

of the soluble carbohydrates ribitol, arabitol, and mannitol in samples of
areolae and marginal prothallus in the lichen
Rhizocarpon geographicum (*** P < 0.001)
Carbohydrate Marginal prothallus Central areolae t

Ribitol 1.29 (0.25) 10.81 (2.13) 4.41***
Arabitol 4.21 (0.70) 16.76 (1.86) 6.31***
Mannitol 3.12 (0.55) 10.21 (1.80) 4.11***
The soluble carbohydrates identified in the samples of R. geographicum were

similar to those recorded previously in Trebouxia containing lichens (Lewis and
Smith, 1967; Richardson et al., 1968; Farrar, 1973; Armstrong and Smith, 1987;
Honegger et al., 1993; Chapman, et al., 1994). Honegger et al. (1993) studied
carbohydrates in 11 cultured lichen fungi and found that their polyol content was
approximately 1% dry weight or less in all species except for Xanthoria parietina
(L.) Th. Fr. and Gyalecta jenensis (Batsch) Zahlbr. In addition to mannitol and
arabitol, which were found in all species; glycerol, volemittol, and erythrytol were
present in some species but the transport carbohydrate ribitol was not recorded in
any of the fungi.
Areolae comprise both algal and fungal tissue while the prothallus consists
almost entirely of fungal tissue. Hence, in the areolae, ribitol should be present
mainly within the algal layer while arabitol and mannitol should be present in
fungal tissue.
Nevertheless, ribitol was consistently recorded in samples taken from the
marginal prothallus (Armstrong and Smith, 1987). The presence of ribitol could
be a consequence of contamination of the prothallus samples by areolae during
collection, the leakage of ribitol from the areolae and reabsorption by the
prothallus, or to the presence of pioneer algal cells or very young areolae
embedded within the marginal prothallus. The low concentration of soluble
carbohydrates within the marginal prothallus suggests that either the prothallus
has a lower demand for carbohydrates or there is restricted transport from areolae
to prothallus. Low concentration of carbohydrate in the prothallus is a possible
explanation for the very low growth rates recorded in this species (Armstrong,
1983; 2005; Armstrong and Smith, 1987; Bradwell and Armstrong, 2007).
VARIATION IN CARBOHYDRATES
BETWEEN SAMPLE TIMES
The levels of ribitol arabitol, and mannitol in areolae and prothallus at each
sample time is shown in Figure 4. Apart from some overall differences in
carbohydrate levels (F = 3.34, P < 0.05), the analysis of variance suggested little
variation in individual carbohydrates between sample days. The ratio of each
carbohydrate in the prothallus to that in the areolae at each sample time is shown
in Figure 5.
The ratios are considerably less than unity for each carbohydrate but vary
through the year. The ratio for mannitol is least in July suggesting particularly low
levels in the prothallus relative to the areolae at this sample time.
Figure 4. Levels of the carbohydrates ribitol, arabitol and mannitol (m per mg extracted
tissue) within the areolae and prothallus of Rhizocarpon geographicum. Analysis of
variance (ANOVA): Sample period F = 3.34 (P < 0.05); Soluble carbohydrate F = 15.72 (P
< 0.001); Sample period x Carbohydrate F = 2.25 (P > 0.05), Prothallus/areolae F = 116.67
(P< 0.001); Prothallus/Areolae x Sample period F = 2.84 (P > 0.05); Prothallus/areolae x
Carbohydrate F = 7.76 (P < 0.01).
Figure 5. The ratios of ribitol, arabitol, and mannitol in the prothallus to areolae in
Rhizocarpon geographicum.
There is some evidence of a change in the ratio of soluble carbohydrates in

prothallus to areolae with sample time. The decline in the ratio especially for
mannitol in July, a period of higher temperatures and less rainfall, suggests its
utilization for stress resistance.
Seasonal variation in carbohydrate levels was suggested by a previous study
of the Trebouxia containing foliose species Xanthoarmelia conspersa (Ehrh. ex
Ach.) Hale (Armstrong and Smith, 1994) in north Wales. In addition, in more
extreme environments, such as the Antarctic, little seasonal variation in
carbohydrates has been recorded although water content of the thallus may vary
seasonally (Melick and Seppelt, 1994). In the study of Montiel (2000), however,
levels of soluble carbohydrates were higher in spring and summer samples of
polar lichens, especially trehalose, which was considered to be involved in low
temperature acclimation and partial dehydration.
CORRELATIONS BETWEEN CARBOHYDRATES

Correlations (Pearsons r) between the concentrations of the three major
carbohydrates ribitol, arabitol, and mannitol in the areolae and prothallus are
shown in Table 3. There was a positive correlation between the concentration of
ribitol in the prothallus and the concentration of mannitol in both the prothallus (r
= 0.72, P < 0.01) and areolae (r = 0.61, P < 0.01). In addition, in the areolae, there
was a positive correlation between the concentrations of ribitol and arabitol (r =
0.87, P < 0.01) and in the prothallus between the concentrations of arabitol and
mannitol (r = 0.77, P < 0.01). A stepwise multiple regression analysis of these
data (Table 4) confirm these results. Hence, concentration of ribitol in the
prothallus is related to two variables, viz., concentration of mannitol both in the
prothallus (F = 14.96, P < 0.01) and areolae (F = 7.54, P < 0.05). The
concentration of arabitol in the prothallus is also related to two variables, viz., the
concentrations of mannitol (F = 13.96, P < 0.01) and ribitol (F = 5.45, P < 0.05) in
the prothallus. Similarly, the concentration of mannitol in the prothallus is related
to both ribitol (F = 14.97, P < 0.01) and arabitol (F = 21.71, P < 0.001) in the
prothallus.
Table 3. Correlations (Pearsons r) between the concentrations of ribitol

(R), arabitol (A) and mannitol (M) in the prothallus (H) and areolae (A) of
Rhizocarpon geographicum (** P < 0.01)
RH RA AH AA MH MA
RH - - - - - -
RA 0.02 - - - - -
AH 0.24 -0.15 - - - -
AA -0.13 0.87** 0.05 - - -
MH 0.72** 0.05 0.77** 0.01 - -
MA 0.61** 0.31 -0.04 0.28 0.28 -
The possible interrelationships between the three soluble carbohydrates in the

prothallus and areolae are shown in Figure 6. There were positive correlations
between the concentrations of the three carbohydrates within the prothallus and
within the areolae but little correlation between carbohydrates in the prothallus
with those in the areolae. Only the concentration of ribitol in the prothallus was
significantly correlated with a carbohydrate in the areolae, viz., mannitol. Hence,
the data suggest that if carbohydrate is supplied to the marginal prothallus from
the central areolae through hyphal connections (Armstrong and Smith, 1987;
1994), then carbohydrates are partitioned differently in the prothallus and largely
independently from the areolae.
Table 4. Forward stepwise multiple regression analysis of the concentrations

of ribitol (R), arabitol (A), and mannitol (M) in the marginal prothallus
(MH) of Rhizocarpon geographicum
Y variable X selected SSRED F SSEX R t

Prothallus MMH 51.67 14.96** 51.67 0.72 3.87
(Ribitol) MAR 17.74 7.54* 69.41 0.83 2.74
Prothallus MMH 49.93 13.96** 49.93 0.71 3.74
(Arabitol) RMH 14.79 5.45* 64.72 0.80 2.33
Prothallus RMH 51.67 14.97** 51.66 0.72 3.86
(Mannitol) AMH 30.23 21.71*** 81.90 0.90 4.66
(AR = central areolae, SSRED = Reduction in sums of squares, F = variance ratio,
SSEX = suns of square explained by all variables extracted, R = multiple regression
coefficient, t = Students t, * P < 0.05, ** P < 0.01, *** P < 0.001)
Figure 6. The possible interrelationships between ribitol, arabitol, and mannitol in the
marginal prothallus and central areolae in Rhizocarpon geographicum (R = Ribitol, A =
arabitol, M = Mannitol).
CONCLUSION
The crustose lichen R. geographicum has an unusual thallus structure
consisting of discrete granules (areolae) containing the algal component growing
in association with a non-lichenised fungal prothallus that extends beyond the
areolae to form a marginal ring. R. geographicum grows at substantially lower
rates than foliose species especially in arctic and alpine environments (Armstrong
and Bradwell, 2010). The supply of carbohydrate to the marginal prothallus may
be a limiting factor and although there is some evidence that the prothallus can
use exogenous sources of carbohydrate (Armstrong and Smith, 1996),
concentrations are unlikely to be sufficient to support growth. Poor rates of
translocation from the areolae to the prothallus may explain the slow growth of
this species.
There has been speculation that R. geographicum may represent one of the
most primitive types of lichen. The existence of a marginal, non-lichenised
prothallus appears to be important to the survival of this species. If the existing
prothallus is removed, a new prothallus is developed within a year, regenerating
first by retreat of the marginal areolae and then by new prothallus growth
(Armstrong and Smith, 1987). It is possible that this growth form is actually an
adaptation to ensure slow growth and consequently, a lower demand for nutrients
from the environment. As a result, a greater concentration of the products of
photosynthesis, such as mannitol, can be allocated to stress resistance rather than
growth thus enabling Rhizocarpon to colonise more extreme environments than
most foliose species.
REFERENCES
Armstrong, RA. The descriptive ecology of saxicolous lichens in an area of South
Merionethshire, Wales. Journal of Ecology 1974; 62: 33-45.
Armstrong RA. Growth curve of the lichen Rhizocarpon geographicum. New
Phytologist 1983; 94: 619-622.
Armstrong RA. The effect of rock surface aspect on growth, size structure and
competition in the lichen Rhizocarpon geographicum. Environmental and
Experimental Botany 2002; 48: 187-194.
Armstrong RA. Radial growth of Rhizocarpon section Rhizocarpon lichen thalli
over six years at Snoqualmie Pass in the Cascade Range, Washington State.
Arctic, Antarctic, & Alpine Research 2005; 37: 411-415.
Armstrong RA. The biology of the crustose lichen Rhizocarpon geographicum.

Symbiosis 2011; 55: 53-68.
Armstrong RA, Bradwell T. Variation in hypothallus width and the growth of the
lichen Rhizocarpon geographicum (L.) DC. Symbiosis 2001; 30: 317-328.
Armstrong RA, Smith SN. Development and growth of the lichen Rhizocarpon
geographicum. Symbiosis 1987; 3: 287-300.
Armstrong RA, Smith SN. The levels of ribitol, arabitol, and mannitol in
individual lobes of the lichen Parmelia conspersa (Ehrh. ex Ach.)Ach.
Environmental and Experimental Botany 1994; 34: 253-260.
Armstrong RA, Smith SN. Do the lichens Xanthoparmelia conspersa (Ach.)Hale
and Rhizocarpon Ram. em Th. Fr. Subgenus Rhizocarpon utilize exogenous
carbohydrates for radial growth? Environmental and Experimental Botany
1996; 36: 13-20.
Armstrong RA, Smith SN. Carbohydrates in the hypothallus and areolae of the
crustose lichen Rhizocarpon geographicum (L.) DC. Symbiosis 2009; 49: 95-
100.
Armstrong RA, Bradwell T. Growth of crustose lichens: A review. Geografiska
Annaler, Series A, Physical Geography 2010; 92A: 3-17.
Asta J & Letrouit-Galinou MA. Observations on the early growth of Rhizocarpon
geographicum thalli. Herzogia 1995; 11: 239-252.
Benedict JB. Techniques in lichenometry: identifying the yellow rhizocarpons.
Arctic and Alpine Research 1988; 22: 244-254.
Beschel RE. Lichenometrical studies in West Greenland. Arctic 1958; 11: 254.
Bradwell T, Armstrong RA. Growth rates of Rhizocarpon geographicum lichens:
a review with new data from Ireland. Journal of Quaternary Science 2007;
22: 311-320.
Chapman BE, Roser DJ, Seppelt RD. C-13NMR analysis of Antarctic cryptogam
extracts. Antarctic Science 1994; 6: 295-305.
Clayden SR. Thallus initiation and development in the lichen Rhizocarpon
lecanorinum. New Phytologist 1998; 139: 685-695.
Dudley SA, Lechowitz MJ. Losses of polyol through leaching in subarctic
lichens. Plant Physiology 1987; 83: 813-815.
Farrar JF. Lichen Physiology: Progress and Pitfalls. In: B.W. Ferry, M.S.
Baddeley and D.L. Hawksworth, eds., Air Pollution and Lichens., pp 238-
282, Athlone Press, University of London, London, 1973.
Farrar JF. Physiological buffering. In Galun M ed., Handbook of Lichenology II,
pp 101-105, CRC Press, Boca Raton, Florida, 1988.
Farrar JF, Smith DC. Ecological physiology of the lichen Hypogymnia physodes
III. The importance of the rewetting phase. New Phytologist 1976; 77: 115-
125.
Hausmann EH. Measurements of the annual growth rate of two species of rock
lichens. Bulletin of the Torrey Botanical Club 1948; 75: 116-117.
Haworth LA, Calkin PE, Ellis JM. Direct measurement of lichen growth in the
central Brooks Range, Alaska, USA, and its application to lichenometric
dating. Arctic & Alpine Research 1986; 18: 289-296.
Honegger R. The lichen symbiosis: What is spectacular about it? Lichenologist
1998; 30: 193-212.
Honegger R., Kutasi V, Ruffner HP. Polyol patterns in 11 species of
aposymbiotically cultured lichen mycobionts. Mycological Research 1993;
97: 35-39.
Holligan M. Routine analysis by gas-liquid chromatography of soluble
carbohydrates in extracts of plant tissue. I. A review of techniques used for
the separation, identification, and estimation of carbohydrates by gas-liquid
chromatography. New Phytologist 1971; 70: 239-269.
Holligan M, Drew EA. Routine analysis by gas-liquid chromatography of soluble
carbohydrates in extracts of plant tissue. II. Quantitative analysis of standard
carbohydrates and separation and estimation of soluble sugars and polyols
from a variety of plant tissues. New Phytologist 1971; 70: 271-297.
Hooker, TN. Lobe growth and marginal zonation in crustose lichens.
Lichenologist 1980; 12: 313-323.
Innes JL. Lichenometry. Progress in Physical Geography 1985; 9: 187-154.
James PW, Hawksworth DL, Rose F. Lichen communities in the British Isles: A
preliminary conspectus. In: M.R.D. Seaward, ed., Lichen Ecology, pp. 295-
419, Academic Press, New York, 1977.
Lewis DH, Smith DC. Sugar alcohols (polyols) in fungi and green plants. I.
Distribution, physiology and metabolism. New Phytologist 1967; 66: 143-
184.
Macfarlane JD, Kershaw KA. Some aspects of carbohydrate metabolism in
lichens. In DH Brown, ed., Lichen Physiology and cell biology, pp 1-8,
Plenum Press, New York, (1985).
Matthews JA. Lichenometric dating: A review with particular reference to 'Little
Ice Age' moraines in southern Norway. In Beck, C. (ed.), Dating in Surface
Context, pp 185-212, Albuquerque, New Mexico Press, 1994.
McCarthy DP. Estimating lichenometric ages by direct and indirect measurement
of radial growth: A case study of Rhizocarpon agg. At the Illecillewaet
Glacier, British Columbia. Arctic Antarctic & Alpine Research 2003; 35: 203-
213.
Melick DR, Seppelt RD. Seasonal investigations of soluble carbohydrates and
pigment levels in Antarctic bryophytes and lichens. Bryologist 1994; 97: 13-
19.
Montiel PO. Soluble carbohydrates (trehalose in particular) and cryoprotection in
polar biota. Cryo-Letters 2000; 21: 83-90.
Mukhtar A, Garty J, Galun M. Does the lichen alga Trebouxia occur free-living in
nature: further immunological evidence. Symbiosis 1994; 17: 247-253.
Nienburg W. 1926. Anatomie der Flechten. K. Linstauerv ed., In: Handbuch der
Pflanzenanatomie. Vol. 6, pp 1-137, , Berlin, Borntraeger, 1926.
Poelt J. Rhizocarpon Ram. em. Th. Fr. subgenus Rhizocarpon in Europe. Arctic &
Alpine Research 1988; 20: 292-298.
Proctor MCF. Sizes and growth rates of thalli of the lichen Rhizocarpon
geographicum on the moraines of the Glacier de Valsorey, Valais,
Switzerland. Lichenologist 1983; 15: 249-261.
Purvis OW, Coppins BJ, Hawksworth DL, James PW, Moore DM. The Lichen
Flora of Great Britain and Ireland. Natural History Museum Publication,
London, 1992.
Richardson DHS, Hill DJ, Smith DC. Lichen physiology XI. The role of the alga
in determining the pattern of carbohydrate movement between lichen
symbionts. New Phytologist 1968; 67: 469-486.
Rogerson RJ, Evans DJA, McCoy WD. Five-year growth of rock lichens in a low-
arctic mountain environment, Northern Labrador. Geog Phys Quatern 1986;
XL: 85-91.
Sancho LG, Pintado A. Evidence of high annual growth rate for lichens in the
martime Antarctic. Polar Biology 2004; 27: 312-319.
Slocum RD, Admadjian V, Hildreth KC. Zoosporogenesis in Trebouxia
gelatinosa: untrastructure, potential for zoospore release and implications for
the lichen association. Lichenologist 1980; 12: 173-187.
Smith DC, Douglas AE. The Biology of Symbiosis. Contemporary Biology,
Edward Arnold, London, 1987.
Whipps J, Cooke RC. Comparative physiology of Albugo tragopogonis-infected
and Puccinia laganophorae-infected plants of Senecio squalidus L. New
Phytologist 1978; 81: 307-319.
Winchester V, Chaujar RK. Lichenometric dating of slope movements, Nant
Ffrancon, North Wales. Geomorphology 2002; 47: 61-74.
In: Mannitol ISBN: 978-1-62808-762-8
Chapter 3
MANNITOL: DISEASE-RELATED CHANGES

AND THEIR USE FOR CLINICAL DISORDERS
David Caldern Guzmn1, Gerardo Barragn Meja1,

Hugo Jurez Olgun2 and Ernestina Hernndez Garca2
1
Neurochemistry Laboratory, National Institute of Pediatrics,
Mexico City, Mexico
2
Pharmacology Laboratory, National Institute of Pediatrics,
Mexico City, Mexico
ABSTRACT
Mannitol, a white, crystalline alcohol, is derived from sugar by
reduction. The pathway to obtain mannitol from natural products is through
hydrogenation of fructose, which is formed from either starch or sugar. This
substance is present in a wide variety of natural products, and in almost all
plants. It is used as an osmotic diuretic agent and a weak renal vasodilator.
Aqueous solutions of mannitol are mildly acidic and sometimes such
solutions are used to decrease the pH. Mannitol is clinically used in
osmotherapy to temporarily reduce acute intracranial pressure while waiting
for the definitive treatment. It is also used to treat patients with oliguric renal
failure. Consequently, mannitol increases water and Na+ excretion, thereby
decreasing extracellular fluid volume. It can also be used as a facilitating
agent for transporting drug agents directly into the brain. This paper reviews
the possible mechanisms of mannitol and its metabolite that are involved in
common clinical disorders. Moreover, the analytical techniques and
42 D. Caldern Guzmn, G. Barragn Meja, H. Jurez Olgun et al.
biochemical markers used to monitor mannitol and its metabolite are

described in this recompilation.
INTRODUCTION
Mannitol is a white, crystalline sugar alcohol. The chemical formula is
C6H8(OH)6 (Figure 1) (table 1). It is used as an osmotic diuretic agent and a weak
renal vasodilator. It was originally isolated from the secretions of flowering ash
and was called manna for its resemblance to the Biblical food [1].
Figure 1. Mannitol.
Table 1. Chemical properties of mannitol
Molecular weight 182.17 g/mol

Density 1.52g/mL
Solubility 25C 22 g/100mL water
Melting point 7.6 torr 165-169 C
Boiling point 3.5 torr 295 C
Mildly acidic 25 C
Relative sweetness of 50 Sucrose = 100
Mannitol is commonly formed via hydrogenation of fructose from starch or

sugar origin (Figure 2).
NADH NAD+
FRUCTOSE MANNITOL
FRUCTOSE-6-PHOSPHATE
GLUCOSE GLUCOSE-6-PHOSPHATE
Figure 2. Metabolic pathway for the synthesis of mannitol.

Mannitol 43
Although starch is cheaper than sucrose (figure 3), the transformation of

starch is much more complicated. However, it yields syrup containing about 42%
fructose, 52% dextrose, and 6% maltose [2]. The syrup is chromatographically
purified to yield 9095% fructose (figure 4). The fructose is then hydrogenated in
a nickel catalyst to get a mixture of isomers of sorbitol and mannitol.
The proportion of the yield is usually 50:50, although under alkaline reaction
conditions, the mannitol yield can slightly increase [1].
Figure 3. Sucrose.
Figure 4. Fructose.
Mannitol is one of the most abundant energy and carbon storage molecules in
nature, produced by a plethora of organisms, including bacteria, yeasts, fungi,
algae and many plants [3]. D-Mannitol is the predominant carbon compound in
conidiospores of the filamentous fungus, Aspergillus niger, and constitutes 0 to
15% of the dry weight. Mannitol 1-phosphate dehydrogenase and mannitol 1-
phosphate phosphatase form the major metabolic pathway for mannitol
biosynthesis in A. niger. Under stress conditions, mannitol appears to be essential
for the protection of A. niger spores against cell damage [4].
Fermentation by microorganisms is a possible alternative to traditional
industrial synthesis, producing much higher yields of mannitol, with minimal to
no side products. Fructose has been discovered in mannitol metabolic pathway
(the mannitol cycle in fungi) in a type of red algae (Caloglossa leprieurii), and it
is highly possible that other microorganisms employ such pathways [5]. A class of
lactic acid bacteria, labeled heterofermentative because of their multiple
fermentation pathways, converts either three fructose molecules, or two fructose
plus a glucose (figure 5) molecule to two mannitol molecules and one molecule of
lactic acid (figure 6), acetic acid, and carbon dioxide each.
Figure 5. Glucose.
Figure 6. Lactic acid.
Mannitol is present in a wide variety of natural products and in almost all

plants. Therefore, it could be directly extracted from natural products, rather than
through chemical or biological syntheses (figure 7) [6].
GLYCOGEN
GLUCOSE GLUCOSE - 6 - P FRUCTOSE - 6 - P
NADH
MANNITOL - 1 - P
NADH+
FRUCTOSE MANNITOL
Figure 7. Metabolic pathways of mannitol in fungi.

Mannitol 45
Mannitol concentrations of plant exudates can range from 20% in seaweeds to

90% in plane tree. Traditionally, mannitol is extracted by Soxhlet extraction using
ethanol (figure 8), water, and methanol to steam and then hydrolyze the crude
material. D-Mannitol belongs to a large and growing family of crystals with two
polymorphs of D-mannitol, and . When they are grown in the presence of
additives such as polyvinylpyrrolidone (PVP) (figure 9) or D-sorbitol (figure 10),
they form ring-banded spherules composed of handed helical fibrils, where the
helix axes correspond to the radial growth directions [7]. The mannitol is then
recrystallized from the extract which gives a yield of about 18% of the original
natural product. The pharmaceutical mannitol is prepared to 20% for clinical use.
Figure 8. Ethanol.
Figure 9. Polyvinylpyrrolidone.
Figure 10. Sorbitol.
For the past 90 years, mannitol has come to be the most widely used
hyperosmolar solution to treat elevated intracranial pressure. Mannitol is
clinically used in osmotherapy in head traumas as a temporary management to
reduce acutely raised intracranial pressure until more definitive treatment can be
applied [8]. It is also used to treat patients with oliguric renal failure. The
administration is intravenous and the filtration is by the glomeruli of the kidney.
However, its reabsorption in the renal tubules is not possible. This results in
decreased water and Na+ reabsorption due to the osmotic effect it exerts.
Consequently, mannitol increases water and Na+ excretion, thereby decreasing
extracellular fluid volume.
In a recent study of 140 patients hospitalized in a Mexican General Hospital
due to different clinical disorders, nearly 8% of these patients were treated with
mannitol (bottle 250ml in solution 20%), however, ambulatory emergency,
pregnant and newborn patients were not included in the study (table 2),
Mannitol can also be used as a facilitating agent for the transportation of
pharmaceuticals [9], and did not appear to alter the effectiveness of the drugs [10].
The arteries of the bloodbrain barrier (BBB) are much more selective than
normal arteries. Normally, molecules can diffuse into tissues through gaps
between the endothelial cells of the blood vessels. However, what enters the brain
must be much more rigorously controlled. The endothelial cells of the bloodbrain
barrier are connected by tight junctions, and simple diffusion through them is
impossible. For this, the need of active transport that requires energy and that can
only transport molecules whose receptor signals exist in the arterial endothelial
cells.
Mannitol is capable of opening this barrier by temporarily shrinking the
endothelial cells and simultaneously stretches the tight junctions between them
[11]. The combination of sodium and mannitol contributes to establish a higher
osmotic gradient across BBB and, furthermore, the progressive accumulation of
mannitol in the ischemic brain tissue counteracts its therapeutic efficacy on
cerebral edema [12].
Mannitol is commonly used in the circuit prime of a heart lung machine
during cardiopulmonary bypass. The presence of mannitol preserves renal
function during the times of low blood flow and pressure, while the patient is on
bypass. The solution prevents the swelling of endothelial cells in the kidney,
which may have otherwise reduced blood flow to this area and provoke cell
damage. Mannitol is contraindicated in patients with anuria and congestive heart
failure [13] (Table 3).
To clarify the mechanism underlying hyperosmotic-induced renal fibrosis in
renal distal tubule cells, Chiang and cols. [27] showed that hyperosmolarity
significantly enhances the susceptibility to exogenous transforming growth factor
(TGF)-beta1, as mannitol (27.5 mM) significantly enhanced the TGF-beta1-
induced increase in fibronectin levels.
Table 2. Clinical disorders in hospitalized Mexican patients (Hospital of 300 beds)
Men Women
Clinical Diseases Age Clinical Diseases age
Maxillary abscess 66 Anemia 66
Perianal abscess 35 Cerebral aneurism 79
Right suprarenal adenoma 44 Bilateral arthralgia 51
Cervical adenopathy 38 Right leg arthrodesis 73
Appendicitis 39 Right hand arthroplasty 61
Appendicitis 33 Total arthroplasty of right hip 79
Acute appendicitis 48 Total arthroplasty of left shoulder 75
Dehydration of tissue in surgical cleaning 25 Arthroscopy of left knee + medial disk meniscus 17
Renal cancer 55 Asthma 50
Hypovolemic shock 38 Bilateral breast biopsy 60
Septic shock 85 Bronchiolitis 1
Percutaneous surgery of left kidney 51 Colon cancer 69
Radical cystectomy + neobladder 46 Breast cancer and bone metastasis 63
Acute cholecistitis 55 Cancer of the bladder 46
Renauteral cholic 49 Bilateral cerebral carotid 51
Hypertensive crisis 56 Lynphangitis in left leg 56
Ventriculoperitoneal shunt 43 Cerebritis 40
Lumbosacral decompression 73 Cholecystectomy 36
Rotators and tendon clamp tear manguito 46 Nissen cholecystectomy 26
Respiratory distress 1 Cholecystitis 83
Valvular double lesion 52 Alitiasic cholecystitis by steroids 31
Abdominal pain 31 Colon-lumbar in study 19
Abdominal pain in study 58 Coxartrosis 62
Lumbar pain 47 Convulsive crisis 65
Table 2. (Continued)
Men Women
Chest pain 54 Epileptic crisis of origin to determine 81
Chest pain in study 44 Septal deformity of hypertrophy 41
Encephalitis mastitis 65 Dermo lipopexia 55
Encephalitis mastitis 65 Abdominal pain 59
Encephalopathy 74 Abdominal pain 38
Metabolic encephalopathy 61 Abdominal pain 15
Diverticular sickness 79 Abdominal pain 39
Vascular cerebral diseases 60 Abdominal pain 19
Ischemic CVA 82 Abdominal pain 72
Fever 20 Abdominal wall seroma drainage 47
Fever 52 Epilepsy 26
Lumbar FX, Left knee FX 29 Pyloric stenosis 51
Decompensated diabetic gastroenteritis 22 Ischemic CVA 80
Infectious gastroenteritis 1 Cerebral-vascular accident (CVA) 74
Gepi 55 Fever + diarrhea 21
Laparoscopic heminefrectomy 56 Right hinge fracture 61
Hemorrhoids and hemicolectomy 61 Anterior cervical fusion 48
Acute myocardial infarction 57 Anterior cervical fusion 60
Airway infections 67 Gastroenteritis 17
Influenza H1N1 1 Glioma 31
Congestive cardiac insufficiency 66 Hemorrhoids 24
Respiratory insufficiency 85 Abdominal wall embedded hernia 62
Melan vs. Angioma lesion of small intestine 65 Total hysterectomy 49
Lymphoma 26 Regional ileitis 61
Men Women
Left renoureteral lithiasis 45 Respiratory insufficiency 53
Ureteral lithiasis 26 Medullar liberation of C4C5 and C5C6 31
Community acquired pneumonia 3 Ganglio-centinela mastectomy 63
Right pneumonia 53 Ganglio-centinela mastectomy 44
Nasal obstruction 41 Pneumonia 67
Intestinal 75 Pneumonia 84
Intestinal occlusion 75 Basal pneumonia 78
Peritonitis 80 Pneumonitis 32
Plastia of right ankle Achilles tendon 72 Intestinal occlusion 59
Thoracic aorta postoperative dissection 63 Inguinal plasty 62
Open radical prostate 68 Grade 11 polycontuntion strain 25
Molar fracture reduction 53 Open radical prostate 60
Resection of right renal cyst and laparoscopy 73 Sarcoma of the radius 13
Resection of mandibular tumor 24 Leukemoide reaction 39
Prostate RTU 74 Reconstruction of bile ducts 28
Vesicle tumor RTU 67 Gastroesophagic reflux 27
Digestive tube bleeding 63 Ritirectomy in study 59
Lower digestive tube bleeding 48 Left knee meniscal rupture 35
Kaposi sarcoma. Neurosiphilis 47 Lower digestive tube bleeding 72
Gaseous gangrene sepsis. Diabetic foot 42 Secon reconstruction TX 42
Syncope in study 19 Painful abdominal syndrome 63
Abdominal tumor 32 Fever syndrome 33
Mediastinal tumors in study 56 Thyroidectomy 54
Varicocele 66 Thyroidectomy 39
Table 3. Recent studies of mannitol used on clinical disorders
Clinical disorders Uses or effects Route Ref.

Hemodialysis patients Decline blood pressure of patients with kidney disease Parenteral [14]
Brain and lung edema Moderate and severe traumatic brain injury Parenteral [15]
Patients with asthma Promotes effective coughing and stimulates mucociliary Inhaled dry powder [16]
clearance
Cystic fibrosis Lung function and reduces exacerbation frequency Inhaled dry powder [17]
Adult and pediatric asthma Decline fractional exhaled nitric oxide as inflammation biomarker Inhaled dry powder [18]
Non-cystic fibrosis bronchiectasis Hydrate the lungs and restore normal mucociliary clearance Inhaled dry powder [19]
Complications of Abdominal aortic Phlebotomy plus mannitol is more effective treatment Parenteral [20]
surgery
Edema by surgical resection of tumor Steroids plus mannitol and radiotherapy Parenteral [21]
metastatic
Persistent asthma Mannitol was well tolerated in a primary care setting Inhaled dry powder [22]
Bacterial meningitis Mannitol reduce the cerebral edema Parenteral [23]
Chronic obstructive pulmonary disease Inhaled mannitol captures eosinophilia in induced sputum Inhaled dry powder [24]
Traumatic and non-traumatic Mannitol decline intracranial pressure Parenteral [25]
encephalopathies.
Asthma To lung function and symptom assessment to aid in the asthma Inhaled dry powder [26]
Diabetic tubular fibrosis Control of hyperosmolarity Parenteral [27]
Acute liver failure Mannitol did not alter osmolarity Parenteral [28]
Severe traumatic brain injured Mannitol decrease intracranial pressure Parenteral [29]
Recurrent brain tumors to intravenous Mannitol did not alter etoposide's tumoricidal effect Parenteral [30]
etoposide
Coma in severe malaria in adults Mannitol therapy as adjunctive treatment for brain swelling in Parenteral [31]
adult cerebral malaria prolongs coma duration and may be
harmful
Mannitol 51
Specifically, hyperosmolarity induced tyrosine phosphorylation on TGF-

beta RII at 336 residues in a time (0-24 h) and dose (5.5-38.5 mM) dependent
manner and increased the level of TGF-beta RI in a dose- and time-course
dependent manner.
Hyperosmolarity significantly downregulated the expression of an
inhibitory Smad (Smad7), decreased the level of Smurf 1, and reduced
ubiquitination of TGF-beta RI. They showed that hyperosmolarity
significantly increased the half-life and inhibited the protein level of TGF-beta
RI by polyubiquitination and proteasome degradation, and suggested that
hyperosmolarity enhances cellular susceptibility to renal tubular fibrosis by
activating the Smad7 pathway and increasing the stability of type I TGF-beta
receptors by retarding proteasome degradation of TGF-beta RI.
In another study of mannitol on acutely comatose patients with severe
diffuse brain swelling and recent clinical signs of impending brain death who
received a novel high-dose mannitol treatment compared with those who
received conventional-dose mannitol in the emergency room revealed that
treatments with high-dose of mannitol in the emergency room was also
associated with significantly better 6-month clinical outcomes (p < 0.02) with
the best rate of favorable outcomes being 43.5% compared with only 9.5% in
the conventional-dose mannitol group. Indeed, ultra-early high-dose mannitol
administration in the emergency room is the first known treatment strategy to
reverse recent clinical signs of impending brain death, and to improved long-
term clinical outcomes for these patients who have previously been considered
unsalvageable [32].
Adult patients suffer age-related macular degeneration. This disorder can
lead to severe central vision loss that is related to age-associated cumulative
oxidative stress of the human retinal epithelial pigment. Mannitol is reported
to be an antioxidant, and as such may play a role in known mechanisms of
some clinical disorders (table 4).
Mannitol as an antioxidant acts by up-regulating the level of catalase by
increasing oxygen free radical (OFR) levels and lipid peroxide (LP) in dose-
dependent manner which is significantly blunted by this compound [44].
Most cells have inherent volume regulatory increase (RVI) mechanisms to
contest an imposed loss in cell size and the mechanism of osmotic stress in
these cells is as follow: Exposure of immature T-cells to hyperosmotic stress
resulted in a rapid, synchronous, and caspase-dependent apoptosis. Multiple
rounds of osmotic stress followed by recovery of cells in normal media
resulted in the development of a population of cells that were resistant to
osmotic stress induced apoptosis.
Table 4. Studies of mannitol reported as antioxidant properties
Tissue Response Ref.

Human skin Mannitol and high glucose could cause [33]
fibroblasts (HSFs) oxidative stress in HSFs, through hydrogen-
rich medium that decreased the excessive
generation of intracellular O2-
Cultured human Genotoxic effects of particulate matter 2.5 were [34]
lung bronchial significantly blocked by mannitol.
epithelial cells
Red cell of blood Mannitol showed the maximum scavenging [35]
(Modified effect in the formation of hydroxyl radical.
hemoglobin)
Human retinal Mannitol protected hRPE cells against the [36]
pigment epithelium H2O2-induced oxidative stress, which acts
(hRPE) through up-regulating the level of catalase.
Kidney Ischemia/reperfusion of the rat pancreas evokes [37]
immediate renal dysfunction. It oxidant-
antioxidant balance is disturbed, but can be
prevented with mannitol.
Pancreas Ex vivo acute pancreatitis induces acute lung [38]
injury via oxidants/antioxidants imbalance,
which is preventable by mannitol.
Human peripheral Increasing concentrations of mannitol [39]
blood mononuclear significantly enhanced lipopolysaccharide-
cells induced cytokine production.
Experimental head Mannitol increases the use of catalase, an [40]
traumas antioxidant enzyme, and GSH-Px, to reduce
cellular damage by reducing the formation of
MDA.
Ischemia-induced Muscle-reduced glutathione and total [41]
and reperfusion glutathione decreased by 27% and 22%,
with mannitol respectively.
Experimental Hyperosmotic perfusion with mannitol [42]
diabetic hearts increased myocardial HSP90 and catalase.
Platelets of patients D-mannitol, which prevent H2O2- and hydroxyl [43]
with non-insulin- radicals-mediated oxidative stress.
dependent diabetes
mellitus
Mannitol 53
These cells were also resistant to other apoptotic stimuli that were
activated via the intrinsic cell death pathway, while remaining sensitive to
extrinsic apoptotic stimuli. Interestingly, these osmotic stress resistant cells
showed no increase in anti-apoptotic proteins, and released cytochrome C
from its mitochondria following exposure to intrinsic apoptotic stimuli [45].
Mannitol is also the basis of Bronchitol which was developed by the
Australian pharmaceutical company (Pharmaxis) as a treatment for cystic
fibrosis and bronchiectasis [46]. Mannitol is orally inhaled as a dry powder
through what is known as an osmohaler and osmotically draws water into the
lungs to thin the thick, sticky mucus characteristic of cystic fibrosis. This is
intended to make it easier for the sufferer to cough the mucus out during
physiotherapy. The critical characteristic of mannitol is its particle size
distribution. It can also be used to temporarily encapsulate a sharp object while
passing through the venous system [47]. Because mannitol dissolves readily in
blood, it can be used in combination with other drugs (table 5).
An intracarotid injection of high molarity mannitol (1.41.6M) causes the
contents of the artery to be hyperosmotic to the cell. Water leaves the cell and
enters the artery in order to recreate an osmotic equilibrium. This loss of water
causes the cells to shrivel and shrink, stretching the tight junctions between the
cells [61]. The newly formed gap reaches its peak within five minutes after
mannitol injection, and stays widely open for thirty minutes. During this time
span, drugs injected into the artery can easily diffuse through the gaps between
cells directly into the brain [62].
Table 5. Studies of pharmaceutical combinations of mannitol plus

other drugs
Mannitol + drugs Effects Ref.

Mannitol + nelfinavir Increasing the fraction of drug deposited on [48]
the olfactory region of the nasal cavity will
result in increased direct nose-to-brain
transport.
Mannitol + leucine, Use of glycine and/or alanine did not achieve [49]
glycine and alanine similar aerosol performance for powder
formulation on particle size, aerosolisation,
emitted dose and cohesion.
Mannitol + hydroxyethyl This combination with HES 130/0.4 at a ratio [50]
starch (HES) of 1:1 should be avoided during craniotomy.
Mannitol + drugs Effects Ref.

Mannitol + The tautomeric forms for S- and R-omeprazole [51]
omeprazole sodium sodium result in changes in the degree of
isomers crystallinity and are responsible for the
interaction with mannitol and may be directly
related to the difference in terms of
bioavailability.
Mannitol + Peroxisome proliferator-activated receptor- [52]
gemfibrozil alpha (PPARalpha) agonists combined with
mannitol, protect organs from normothermic,
perfusion-induced damage.
Mannitol + N- Co-administration of Mannitol and N- [53]
acetylcysteine acetylcysteine augments the amount of lung
protection afforded by each drug individually
and enhances their antioxidant potentials.
Mannitol + Agents that have been shown to ameliorate [54]
furosemide experimental cisplatin nephrotoxicity.
Mannitol + AM prevented mannitol-induced apoptosis in a [55]
adrenomedullin dose-dependent manner in endothelial cells,
(AM) through phosphatidylinositol 3'-kinase/Akt
pathway.
Mannitol + Furosemide and mannitol both significantly [56]
furosemide suppressed spontaneous epileptic spikes and
electrical stimulation-evoked epileptiform
discharges.
Mannitol + The combination of mannitol and furosemide [57]
furosemide resulted in greater reduction of brain water
content than did mannitol alone.
Mannitol + Fexofenadine reduced sensitivity to mannitol in [58]
fexofenadine subjects with asthma as response leukotriene
mechanism.
Mannitol and steroid Acceptable and effective treatment for Complex [59]
with oral gabapentin regional pain syndrome type 1.
Mannitol + lidocaine Resulted in a statistically higher success rate [60]
with epinephrine (response) in inferior alveolar nerve compared
with formulation without mannitol.
This makes mannitol indispensable for delivering various drugs directly to

the brain (e.g., in chemotherapy for brain tumors) [63]. For this reason, it is
Mannitol 55
important to know safe and confident methods for the measurement of

mannitol (Table 6).
Table 6. Methods for measurement of mannitol in natural products or

biological fluids
Method Sample Ref

Gas chromatography Plants [64]
Gas-liquid chromatography plasma, urine, and bile [65]
Enzymatic method Serum [66]
Enzymatic method Plants [67]
Microplate assay Crude bacterial extracts [68]
Enzymatic method Urine [69]
Liquid chromatography Urine [70]
Gas-liquid chromatography Urine [71]
Liquid chromatography Urine [72]
Gas chromatography Urine [73]
Photometric Urine [74]
Gas chromatography/mass spectrometry- Urine [75]
positive chemical ionization
REFERENCES
[1] Lawson, P. In Mannitol; Blackwell Publishing Ltd: pp 219225, 2007.
[2] Eggleston, G., Yen, J.W., Alexander, C., Gober, J.. Measurement and
analysis of the mannitol partition coefficient in sucrose crystallization
under simulated industrial conditions. Carbohydr. Res. 355, 69-78,
(2012).
[3] Song, S.H., Vieille, C. Recent advances in the biological production of
mannitol. Appl. Microbiol. Biotechnol. 84, 5562, (2009).
[4] Ruijter, G.J., Bax, M., Patel, H., Flitter, S.J., van de Vondervoort, P.J.,
de Vries, R.P., vanKuyk, P.A., Visser, J. Mannitol is required for stress
tolerance in Aspergillus niger conidiospores. Eukaryot. Cell. 2(4), 690-8,
(2003).
[5] Ghoreishi, S.M., Shahrestani, R.G. Innovative strategies for engineering
mannitol production. Trends Food Sci. Technol. 20, 263270, (2009).
[6] Cheng, F.Y., Zamski, E., Guo, W.W., Pharr, D.M., Williamson, J.D.
Salicylic acid stimulates secretion of the normally symplastic enzyme
mannitol dehydrogenase: a possible defense against mannitol-secreting

fungal pathogens. Planta 230(6), 1093-103, (2009).
[7] Shtukenberg, A.G., Cui, X., Freudenthal, J., Gunn, E., Camp, E., Kahr,
B. Twisted mannitol crystals establish homologous growth mechanisms
for high-polymer and small-molecule ring-banded spherulites. J. Am.
Chem. Soc. 134(14), 6354-64, (2012).
[8] Hinson, H.E., Stein, D., Sheth, K.N. Hypertonic saline and mannitol
therapy in critical care neurology. J. Intensive Care Med. 28(1), 3-11,
(2013).
[9] Hernndez, G.E., Caldern, G.D., Jurez, O.H., Trujillo, J.F., Nuez,
A.E., Pierdant, R.F., Barragn, M.G., Labra, R.N., Santamara, D.D.
Effect of cerebrolysin on the levels of glutathione and 5-HT in different
regions of rat brain in presence of dantrolene.
[10] Adi, H., Young, P.M., Chan, H.K., Agus, H., Traini, D. Co-spray-dried
mannitol-ciprofloxacin dry powder inhaler formulation for cystic
fibrosis and chronic obstructive pulmonary disease. Eur. J. Pharm. Sci.
40(3), 239-47, (2010).
[11] Best, B. Perfusion and Diffusion in Cryonics Protocol. Accessed
November 10, 2010
[12] Zeng, H.K., Wang, Q.S., Deng, Y.Y., Jiang, W.Q., Fang, M., Chen,
C.B., Jiang, X. A comparative study on the efficacy of 10% hypertonic
saline and equal volume of 20% mannitol in the treatment of
experimentally induced cerebral edema in adult rats. BMC Neurosci. 11,
153, (2010).
[13] De Vecchis, R., Ciccarelli, A., Pucciarelli, A. Unloading therapy by
intravenous diuretic in chronic heart failure: a double-edged weapon? J.
Cardiovasc. Med. (Hagerstown) 11(8), 571-4, (2010).
[14] McCausland, F.R., Prior, L.M., Heher, E., Waikar, S.S. Preservation of
blood pressure stability with hypertonic mannitol during hemodialysis
initiation. Am. J. Nephrol. 36(2), 168-74, (2012).
[15] Marks, J.A., Li, S., Gong, W., Sanati, P., Eisenstadt, R., Sims, C., Smith,
D.H., Reilly, P.M., Pascual, J.L. Similar effects of hypertonic saline and
mannitol on the inflammation of the blood-brain barrier microcirculation
after brain injury in a mouse model. J. Trauma. Acute Care Surg. 73(2),
351-7, (2012).
[16] Daviskas, E., Rubin, B.K. Effect of inhaled dry powder mannitol on
mucus and its clearance. Expert Rev. Respir. Med. 7(1), 65-75, (2013).
[17] Hurt, K., Bilton, D. Inhaled mannitol for the treatment of cystic fibrosis.
Expert Rev. Respir. Med. 6(1), 19-26, (2012).
Mannitol 57
[18] Barben, J., Strippoli, M.P., Trachsel, D., Schiller, B., Hammer, J.,
Kuehni, C.E. Effect of mannitol dry powder challenge on exhaled nitric
oxide in children. PLoS One 8(1):e54521, (2013).
[19] Gjoerup, J., Hilberg, O., Bendstrup, E. Inhaled mannitol in the treatment
of non-cystic fibrosis bronchiectasis in adults. Respirology 17(6), 927-
32, (2012).
[20] Goksin, I., Adali, F., Enli, Y., Akbulut, M., Teke, Z., Sackan, G., Ocak,
E., Ozcan, A.V. The effect of phlebotomy and mannitol on acute renal
injury induced by ischemia/reperfusion of lower limbs in rats. Ann.
Vasc. Surg. 25(8), 1118-28, (2011).
[21] Shapiro, R.H., Chang, A.L. Urgent radiotherapy is effective in the
treatment of metastatic medulloblastoma causing symptomatic brainstem
edema. Pediatr. Blood Cancer 57(6), 1077-80, (2011).
[22] Lipworth, B.J., Short, P.M., Williamson, P.A., Clearie, K.L., Fardon,
T.C., Jackson, C.M. A randomized primary care trial of steroid titration
against mannitol in persistent asthma: STAMINA trial. Chest 141(3),
607-15, (2012).
[23] Liu, S., Li, L., Luo, Z., Wang, M., She, H., Yu, X., Deng, X., Huang, F.,
Shang, L., Jian, C., Ji, G., Yue, S. Superior effect of hypertonic saline
over mannitol to attenuate cerebral edema in a rabbit bacterial meningitis
model. Crit. Care Med. 39(6), 1467-73, (2011).
[24] de Nijs, S.B., Fens, N., Lutter, R., Dijkers, E., Krouwels, F.H., Smids-
Dierdorp, B.S., van Steenwijk, R.P., Sterk, P.J. Airway inflammation
and mannitol challenge test in COPD. Respir. Res. 12, 11, (2011).
[25] Gwer, S., Gatakaa, H., Mwai, L., Idro, R., Newton, C.R. The role for
osmotic agents in children with acute encephalopathies: a systematic
review. BMC Pediatr. 10, 23, (2010).
[26] Porsbjerg, C., Backer, V., Joos, G., Kerstjens, H.A., Rodriguez-Roisin,
R. Current and future use of the mannitol bronchial challenge in
everyday clinical practice. Clin. Respir. J. 3(4), 189-97, (2009).
[27] Chiang, T.A., Yang, Y.L., Yang, Y.Y., Hu, M.H., Wu, P.F., Liu, S.F.,
Huang, R.M., Liao, T.N., Hung, C.Y., Hung, T.J., Lee, T.C.
Hyperosmolarity enhanced susceptibility to renal tubular fibrosis by
modulating catabolism of type I transforming growth factor-beta
receptors. J. Cell. Biochem. 109(4), 663-71, (2010).
[28] Saraswat, V.A., Saksena, S., Nath, K., Mandal, P., Singh, J., Thomas,
M.A., Rathore, R.S., Gupta, R.K. Evaluation of mannitol effect in
patients with acute hepatic failure and acute-on-chronic liver failure
using conventional MRI, diffusion tensor imaging and in-vivo proton

MR spectroscopy. World J. Gastroenterol. 14(26), 4168-78, (2008).
[29] Gasco, J., Sendra, J., Lim, J., Ng, I. Linear correlation between stable
intracranial pressure decrease and regional cerebral oxygenation
improvement following mannitol administration in severe acute head
injury patients. Acta Neurochir. Suppl. 95,73-7, (2005).
[30] Kobrinsky, N.L., Packer, R.J., Boyett, J.M., Stanley, P., Shiminski-
Maher, T., Allen, J.C., Garvin, J.H., Stewart, D.J., Finlay, J.L. Etoposide
with or without mannitol for the treatment of recurrent or primarily
unresponsive brain tumors: a Children's Cancer Group Study, CCG-
9881. J. Neurooncol. 45(1), 47-54, (1999).
[31] Mohanty, S., Mishra, S.K., Patnaik, R., Dutt, A.K., Pradhan, S., Das, B.,
Patnaik, J., Mohanty, A.K., Lee, S.J, Dondorp, A.M. Brain swelling and
mannitol therapy in adult cerebral malaria: a randomized trial. Clin.
Infect. Dis. 53(4), 349-55, (2011).
[32] Cruz, J., Minoja, G., Okuchi, K., Facco, E. Successful use of the new
high-dose mannitol treatment in patients with Glasgow Coma Scale
scores of 3 and bilateral abnormal pupillary widening: a randomized
trial. J. Neurosurg. 100(3), 376-83, (2004).
[33] Yu, P., Wang, Z., Sun, X., Chen, X., Zeng, S., Chen, L., Li, S.
Hydrogen-rich medium protects human skin fibroblasts from high
glucose or mannitol induced oxidative damage. Biochem. Biophys. Res.
Commun. 409(2), 350-5, (2011).
[34] Oh, S.M., Kim, H.R., Park, Y.J., Lee, S.Y., Chung, K.H. Organic
extracts of urban air pollution particulate matter (PM2.5)-induced
genotoxicity and oxidative stress in human lung bronchial epithelial cells
(BEAS-2B cells). Mutat. Res. 723(2), 142-51, (2011).
[35] Khaket, T.P., Ahmad, R. Biochemical studies on hemoglobin modified
with reactive oxygen species (ROS). Appl. Biochem. Biotechnol. 164(8),
1422-30, (2011).
[36] Liu, J.H., Chen, M.M., Huang, J.W., Wann, Ho, L.K., Pan, W.H., Chen,
Y.C., Liu. C.M., Yeh, M.Y., Tsai, S.K., Young, M.S., Ho, L.T., Kuo,
C.D., Chuang, H.Y., Chao, F.P., Chao, H.M. Therapeutic effects and
mechanisms of action of mannitol during H2O2-induced oxidative stress
in human retinal pigment epithelium cells. J. Ocul. Pharmacol. Ther.
26(3), 249-57, (2010).
[37] Khoury, W., Namnesnikov, M., Fedorov, D., Abu-Gazala, S.,
Weinbroum, A.A. Mannitol attenuates kidney damage induced by
Mannitol 59
xanthine oxidase-associated pancreas ischemia-reperfusion. J. Surg. Res.

160(1), 163-8, (2010).
[38] Weinbroum, A.A. Mannitol prevents acute lung injury after pancreas
ischemia-reperfusion: a dose-response, ex vivo study. Lung 187(4), 215-
24, (2009).
[39] Otto, N.M., Schindler, R., Lun, A., Boenisch, O., Frei, U., Oppert, M.
Hyperosmotic stress enhances cytokine production and decreases
phagocytosis in vitro. Crit. Care 12(4), R107, (2008).
[40] Yilmaz, N., Dulger, H., Kiymaz, N., Yilmaz, C., Gudu, B.O., Demir, I.
Activity of mannitol and hypertonic saline therapy on the oxidant and
antioxidant system during the acute term after traumatic brain injury in
the rats. Brain Res. 1164, 132-5, (2007).
[41] Westman, B., Weidenhielm, L., Rooyackers, O., Fredriksson, K.,
Wernerman, J., Hammarqvist, F. Knee replacement surgery as a human
clinical model of the effects of ischaemia/reperfusion upon skeletal
muscle. Clin. Sci. (Lond) 113(7), 313-8, (2007).
[42] Chen, H., Shen, W.L., Wang, X.H., Chen, H.Z., Gu, J.Z., Fu, J., Ni,
Y.F., Gao, P.J., Zhu, D.L., Higashino, H. Paradoxically enhanced heart
tolerance to ischaemia in type 1 diabetes and role of increased
osmolarity. Clin. Exp. Pharmacol. Physiol. 33(10), 910-6, (2006).
[43] Jardn, I., Redondo, P.C., Salido, G.M., Pariente, J.A., Rosado, J.A.
Endogenously generated reactive oxygen species reduce PMCA activity
in platelets from patients with non-insulin-dependent diabetes mellitus.
Platelets 17(5), 283-8, (2006).
[44] Liu, J.H., Chen, M.M., Huang, J.W., Wann, H., Ho, L.K., Pan, W.H.,
Chen, Y.C., Liu, C.M., Yeh, M.Y., Tsai, S.K., Young, M.S., Ho, L.T.,
Kuo, C.D., Chuang, H.Y., Chao, F.P., Chao, H.M. Therapeutic effects
and mechanisms of action of mannitol during H2O2-induced oxidative
stress in human retinal pigment epithelium cells. J. Ocul. Pharmacol.
Ther. 26(3), 249-57, (2010).
[45] Bortner, C.D., Scoltock, A.B., Sifre, M.I., Cidlowski, J.A. Osmotic
stress resistance imparts acquired anti-apoptotic mechanisms in
lymphocytes. J. Biol. Chem. 287(9), 6284-95, (2012).
[46] Teper, A., Jaques, A., Charlton, B. Inhaled mannitol in patients with
cystic fibrosis: A randomised open-label dose response trial. J. Cyst
Fibros 10(1), 1-8, (2011).
[47] Parkerson, J., Ledford, D. Mannitol as an indirect bronchoprovocation
test for the 21st century. Ann. Allergy Asthma Immunol. 106(2), 91-6,
(2011).
[48] Hoekman, J.D., Ho, R.J. Effects of localized hydrophilic mannitol and
hydrophobic nelfinavir administration targeted to olfactory epithelium
on brain distribution. AAPS Pharm. Sci. Tech. 12(2), 534-43, (2011).
[49] Sou, T., Orlando, L., McIntosh, M.P., Kaminskas, L.M., Morton, D.A.
Investigating the interactions of amino acid components on a mannitol-
based spray-dried powder formulation for pulmonary delivery: A design
of experiment approach. Int. J. Pharm. 421(2), 220-9, (2011).
[50] Lindroos, A.C., Schramko, A., Tanskanen, P., Niemi, T. Effect of the
combination of mannitol and ringer acetate or hydroxyethyl starch on
whole blood coagulation in vitro. J. Neurosurg. Anesthesiol. 22(1), 16-
20, (2010).
[51] Agatonovic-Kustrin, S., Markovic, N., Ginic-Markovic, M., Mangan,
M., Glass, B.D. Compatibility studies between mannitol and omeprazole
sodium isomers. J. Pharm. Biomed. Anal. 48(2), 356-60, (2008).
[52] Jackson, T.C., Mi, Z., Bastacky, S.I., McHale, T., Melhem, M.F.,
Sonalker, P.A., Tofovic, S.P., Jackson ,E.K. PPAR alpha agonists
improve renal preservation in kidneys subjected to chronic in vitro
perfusion: interaction with mannitol. Transpl. Int. 20(3), 277-90, (2007).
[53] Weinbroum, A.A. Concomitant administration of mannitol and N-
acetylcysteine for the prevention of lung reperfusion injury. J. Trauma
60(6), 1290-6, (2006).
[54] Ali, B.H., Al, Moundhri, M.S. Agents ameliorating or augmenting the
nephrotoxicity of cisplatin and other platinum compounds: a review of
some recent research. Food Chem. Toxicol. 44(8), 1173-83, (2006).
[55] Kim, W., Moon, S.O., Sung, M.J., Kim, S.H., Lee, S., Kim, H.J., Koh,
G.Y., Park, S.K. Protective effect of adrenomedullin in mannitol-
induced apoptosis. Apoptosis 7(6), 527-36, (2002).
[56] Haglund, M.M., Hochman, D.W. Furosemide and mannitol suppression
of epileptic activity in the human brain. J. Neurophysiol. 94(2), 907-18,
(2005).
[57] Thenuwara, K., Todd, M.M., Brian, J.E.Jr. Effect of mannitol and
furosemide on plasma osmolality and brain water. Anesthesiology 96(2),
416-21, (2002).
[58] Brannan, J.D., Anderson, S.D., Gomes, K., King, G.G., Chan, H.K.,
Seale, J.P. Fexofenadine decreases sensitivity to and montelukast
improves recovery from inhaled mannitol. Am. J. Respir. Crit. Care
Med. 163(6), 1420-5, (2001).
Mannitol 61
[59] Lee, S.K., Yang, D.S., Lee, J.W., Choy, W.S. Four treatment strategies
for complex regional pain syndrome type 1. Orthopedics 35(6), 834-42,
(2012).
[60] Kreimer, T., Kiser, R., Reader, A., Nusstein, J., Drum, M., Beck, M.
Anesthetic efficacy of combinations of 0.5 mol/L mannitol and lidocaine
with epinephrine for inferior alveolar nerve blocks in patients with
symptomatic irreversible pulpitis. J. Endod. 38(5), 598-603, (2012).
[61] Ikeda, M., Bhattacharjee, A.K., Kondoh, T., Nagashima, T., Tamaki, N.
Synergistic Effect of Cold Mannitol and Na+/Ca2+ Exchange Blocker
on Blood-Brain Barrier Opening. Biochem. Biophys. Res. Commun. 291,
669674, (2002).
[62] Wang, M., Etu, J., Joshi, S. Enhanced disruption of the blood brain
barrier by intracarotid mannitol injection during transient cerebral
hypoperfusion in rabbits. J. Neurosurg. Anesthesiol. 19, 249256,
(2007).
[63] Miyagami, M., Tsubokawa, T., Tazoe, M., Kagawa, Y. Intra-arterial
ACNU chemotherapy employing 20% mannitol osmotic blood-brain
barrier disruption for malignant brain tumors. Neurol. Med. Chir.
(Tokyo) 30(8), 582-90, (1990).
[64] Fritz, M., Ehwald, R. Mannitol permeation and radial flow of water in
maize roots. New Phytol, 189(1), 210-7, (2011).
[65] Laker, M.F., Mount, J.N. Mannitol estimation in biological fluids by
gas-liquid chromatography of trimethylsilyl derivatives. Clin. Chem.
26(3), 441-3, (1980).
[66] Blomquist, C.H., Snyder, B.D., Niehaus, W.G. Improved enzymatic
method for determining mannitol and its application to dog serum after
mannitol infusion. J. Clin. Chem. Clin. Biochem. 19(3), 139-43, (1981).
[67] Dills, W.L.Jr, Geller, J.I., Gianola, K.M., McDonough, G.M. Enzymatic
analysis of 2,5-anhydro-D-mannitol and related compounds. Anal.
Biochem. 133(2), 344-9, (1983).
[68] Mills, J., Allison, N. A rapid quantitative microplate assay for NAD-
linked D-mannitol dehydrogenase. Lett. Appl. Microbiol. 11(4), 211-3,
(1990).
[69] Blood, J., Ingle, A.R., Allison, N., Davies, G.R., Hill, P.G. Rapid
enzymatic method for the measurement of mannitol in urine. Ann. Clin.
Biochem. 28(Pt 4), 401-6, (1991).
[70] Willems, D., Cadranel, S., Jacobs, W. Measurement of urinary sugars by
HPLC in the estimation of intestinal permeability: evaluation in pediatric
clinical practice. Clin. Chem. 39(5), 888-90, (1993).
[71] Blomquist, L., Bark, T., Hedenborg, G., Svenberg, T., Norman, A.
Comparison between the lactulose/mannitol and 51Cr-
ethylenediaminetetraacetic acid/14C-mannitol methods for intestinal
permeability. Frequency distribution pattern and variability of markers
and marker ratios in healthy subjects. Scand. J. Gastroenterol. 28(3),
274-80, (1993).
[72] Fleming, S.C., Duncan, A., Russell, R.I., Laker, M.F. Measurement of
sugar probes in serum: an alternative to urine measurement in intestinal
permeability testing. Clin. Chem. 42(3), 445-8, (1996).
[73] Zhu, X., Xiong, D., Sheng, Z. Measurement of urinary content of
lactulose and mannitol by gas chromatography as an index of
permeability of the gut. Zhonghua Wai Ke Za Zhi 35(4), 248-50, (1997).
[74] Graefe, H., Gtschow, B., Gehring, H., Dibbelt, L. Sensitive and specific
photometric determination of mannitol in human serum. Clin. Chem.
Lab. Med. 41(8), 1049-55, (2003).
[75] Lee, J., Chung, B.C. Simultaneous measurement of urinary polyols using
gas chromatography/mass spectrometry. J. Chromatogr. B Analyt.
Technol. Biomed. Life Sci. 831(1-2), 126-31, (2006).
In: Mannitol ISBN: 978-1-62808-762-8
Chapter 4
USE OF MANNITOL IN THERMAL ENERGY

STORAGE APPLICATIONS
Luisa F. Cabeza1, Camila Barreneche1,2, Antoni Gil1

and A. Ins Fernndez2
1
GREA Innovaci Concurrent, Universitat de Lleida,
Edifici CREA, Lleida, Spain
2
Department of Materials Science & Metallurgical Engineering,
Universitat de Barcelona, Barcelona, Spain
CIC Energigune, Miano, lava, Spain
ABSTRACT
Nowadays thermal energy storage (TES) systems are proposed as
one of the most powerful technologies to be charged with heat (or cold)
and hold energy over time by shifting demand over time to reduce peak
loads and facilitating the greater use of renewable energy by storing the
energy produced so it can coincide with demand. TES systems are able to
store energy as sensible heat leading with temperature increment of the
storage medium, as latent heat storing energy using the latent heat
produced when a phase change state occur using phase change materials
PCM, and as chemical reaction energy storing energy using a
exothermic/endothermic reversible reaction by thermochemical materials
(TCM). Solar cooling and air-conditioning is a technology that allows
Tel: +34.973.00.35.76. Email: lcabeza@diei.udl.cat.

64 Luisa F. Cabeza, Camila Barreneche, Antoni Gil et al.
coincidence of solar gains with cooling loads reducing peak loads created
by air-conditioning. TES systems can be coupled between absorption
chillers and solar collectors in order to use the energy stored when the
there is a peak load or the system is practically discharged. In addition,
phase change materials PCM candidates must fulfill several conditions
to be used as storage materials: melting point of PCM must be closed to
selected work temperature range, high latent heat and high specific heat,
elevate thermal conductivity (solid and liquid state) to support charging
and discharging processes inside the storage system. Additionally, the
change volume during phase change transformation must be minimum, as
well as the pressure vapor, allowing the use of conventional containers.
Moreover, it must melt congruently with minimum subcooling and it
must be chemically stable. D-mannitol has a phase change temperature at
167 C and a phase change enthalpy is around 316 kJkg-1. These
thermophysical properties turn d-mannitol as a perfect candidate to be
used as PCM and it was studied with this purpose. D-mannitol was
characterized performing differential scanning calorimetry under dynamic
mode using a 0.5 Kmin-1 heating rate between 25 C and 200 C. This
substance was cycled several times and results shows 3 different thermal
behavior: The first one was a single peak at 167 C, the second has
double peak at 156 C and 167 C, and the third thermal behavior is a
single peak at 157 C. Accordingly, there is a polymorphic transformation
which was studied with FT-IR and two different phases were identified:
-phase and -phase. Then, a temperature range was established to work
with this substance as PCM between 135 C and 175 C. This working
range includes the phase change transformation of the two phases under
analysis ( and ). To test the d-mannitol thermal behaviour at pilot plant
scale, 150 kg of d-mannitol were introduced in a storage tank which was
designed as shell-and-tubes heat exchanger. Results show that applying
different cooling conditions produces d-mannitol polymorphic changes.
Moreover, it has been shown that the working range (between 135C and
175C) is adequate for pilot plant experiments.
1. INTRODUCTION
Energy efficiency and generation are key points in the development of
countries and these factors have direct impact with their own inner production
which is priority important as well considering both emerging economies and
the countries of the OECD (Organization for Economic and Co-operation and
Development). Currently, energy demand has increased dramatically; making
OECD countries to invest a part of their budgets for research and development
to create new alternatives to the adjustment of energy demand and generation.
Use of Mannitol in Thermal Energy Storage Applications 65
Figure 1. Fresnel solar panels for solar energy harvesting.
Due to the current high price of oil and the increase of CO2 emissions,
unrivalled alternative is renewable energy, which do not involve any
environmental impact when they are implement to generate electricity or
energy.
Leading renewable energy is consolidated solar energy to heat domestic
hot water (DHW) via thermal panels or allows the generation of electricity by
installing photovoltaic panels locally (in a house/building) or in large solar
plants generating large amount of energy (see Figure 1). The foremost
advantage of solar energy is its low cost (it is the cheapest renewable energy)
and widespread availability.
However, solar energy presents a drawback which is not easily solvable:
this renewable energy is time depending and in some applications this fact
produces a mismatch between energy demand and energy supply.
A possible solution to this drawback raised above is the installation of
thermal energy storage (TES) systems. These systems are able to store the
excess energy produced by charging different materials and discharging them
during peaks of energy demand or when it is necessary.
1.1. Thermal Energy Storage (TES)
Thermal energy storage (TES) will play a key role in the successful
application of thermal heating and cooling technologies. Meanwhile, the
International Energy Agency (IEA) suggests energy storage as a possible
solution for the energy problems described, often focusing on materials and
systems for building and industrial applications.
Thermal energy can be stored following three processes: as sensible heat,
as latent heat or as thermochemical energy. Furthermore, TES systems can be
charged with heat (or cold) and hold energy over time by shifting demand and
reducing the peak loads. These facts will facilitate the greater use of renewable
energy.
Abhat et al. [1] proposed in 1983 a classification of materials to be used as
thermal energy storage materials which is shown in Figure 2.
Figure 2. Classification of substances used as PCM for thermal energy storage [1].
1.1.1. Thermal Energy Storage As Sensible Heat

The heat transferred to a storage medium leads to a temperature increase
of that medium. The heat stored in this process is known as sensible heat [2]
and this process can be achieved with solid, liquid or gas material states. Then,
the energy stored by one substance or material as sensible heat is analyzed
when it is subject to temperature changes taking into account its specific heat.
Figure 1 shows the thermophysical behaviour observed for one material or
substance storing energy as sensible heat. The most common material used to
store energy as sensible heat is water which has a specific heat capacity of
4.18 kJkg-1K-1. Sensible heat storage has two main advantages: it is cheap
and without the risks derived from the use of toxic materials.
Heat capacity is given from content of material used, volume or mass.
Sensible heat is often used with solids like stone or brick, or liquids as water
(high specific heat as mentioned).
Temperature (C)
Stored heat
Figure 3. Thermal energy storage profile vs. temperature when heat is stored as
sensible heat.
1.1.2. Thermal Energy Storage As Latent Heat Phase Change

Materials
Thermal energy storage (TES) proposes phase change material (PCM) as
materials capable of storing a high quantity of energy as latent heat during the
phase change. PCM are presented as an option to increase the thermal mass of
building envelopes and building systems. Such materials have been
extensively studied in the past by renowned researchers [2-7]. Among all
materials, those that have high storage density for small temperature range are
considered PCM [8] and PCM are classified as different groups depending on
the material nature.
Conventional profile when one PCM is submitted to a temperature
increment is shown in Figure 4. A temperature increment produces a
temperature increase on the material evaluated. Then, the temperature remains
constant during the phase change occurring but heat stored is increased.
Further transfer of heat results as sensible heat again when the phase change is
finished.
Temperature (C)
Stored heat
Figure 4. Thermal energy storage profile vs. temperature when heat is stored as latent.
1.1.3. Thermochemical Energy Storage

Thermochemical materials (TCM) are materials which can store energy by
a reversible endothermic/exothermic reaction/process and the resulting
reaction products are easily separated (usually a gassolid system or a liquid
solid system) [9]. This storage process is being hard investigated recently in
order to obtain a compact system for seasonal or long term storage [9].
CHARGE
STORE
DISCHARGE
Figure 5. Steps followed by TCM to store energy.

First of all, it is performed the charging process: applying heat, the

material reacts and it is separated in two parts: A + B. Then, storing process
takes place: the heat is stored because the products are easily separated and the
storage could be complete at low temperature. Finally, the discharging process
is performed: as a reversible reaction, when products A + B are placed
together and under the suitable conditions to react, the energy is released
again. This process is shown in Figure 5.
1.2. Solar Cooling Application
Thermal energy storage (TES) systems are nowadays considered as an

appreciative solution for the time dependency of solar energy. When solar
energy is used in solar water absorption systems, sun power is considered the
heat input for the generator installed in the system but this heat source must be
constant. Throughout the day, there may be a scenario where there is heat
surplus that should not be transmitted to the generator. TES system at this time
may be proper solution since it can handle the fact that the heat source is not
constant (solar energy) providing the requirement of solar cooling storage
system.
Thereby, heat surplus circulate by the heat transfer fluid (HTF) through
the thermal storage system which contains a cold PCM (solid state). This solid
is able to store high amount of energy and it is heat up progressive since it
starts to melt. At this moment, latent heat is being used to store the energy
coming from the Fresnel collectors or solar collectors. This storage system is
holding back the heat input generated by these collectors.
Later on, when the heat source is not hot enough and HTF temperature
decrease, heat stored by TES system must be used in order to increase the HTF
temperature. Hence, heat input remains constant and HTF will provide the heat
input needed by the generator to work properly.
Therefore, TES was extensively assumed by the international scientist
community as a solution to reduce peak loads and for the energy savings and
energy efficiency [11-12].
Furthermore, PCM have been widely studied as the material used to be
include in TES system because these materials presents a temperature working
range variable depending on the material which can be adjustable to the
application requirements.
The phase change temperature required for solar cooling application is

between 140 C and 200 C being the minimum or maximum temperature
input from solar collectors, respectively [13].
Moreover, the TES vessel is located between solar/Fresnel collectors and
absorption chiller as the scheme presented in Figure 6 shows.
Figure 6. Location of thermal energy storage tank containing the PCM
1.3. Materials to Be Used as PCM for TES in Solar Cooling

Applications
TES systems for solar cooling work between 140 C and 200 C and
materials to be used as PCM for this application must own a phase change
temperature close to this temperature range. Furthermore, another important
requirement is the storage capacity: the PCM selected has to perform the
highest heat storage capacity through the highest latent heat of fusion.
Materials with this requirement found in the literature are listed in Table 1
[5-7].
Table 1 shows that the material with highest phase change enthalpy and
with a suitable melting temperature for this application is d-mannitol. Thereby,
it is a potential candidate to be used as PCM for thermal energy storage in
solar cooling applications.
This material present several polymorphic phases and thermophysical
properties of these phases are slightly different. This information is listed in
Table 2. All phases have the temperature of phase change within the
application temperature range.
Table 1. PCM candidates to be used for solar cooling applications [5-7]
Material Phase change temperature Phase change enthalpy

(C) (kJkg-1)
Salicylic acid 159 199
Benzanilide 161 162
D-mannitol 167 316
Hydroquinone 172.4 258
Potassium thiocyanate 173 280
Table 2. Melting temperature of d-mannitol phases [14]
D-mannitol phases Melting temperature (C)

phase 157
phase 167
phase 155
2. POLYMORPHISM AT LABORATORY
Thermophysical properties behavior of PCM must be studied previously
of the implementation of one material to be used in one application. In fact,
this is a key point and has to be accomplished before the putting this material
into practice.
One of the most powerful technics to characterize the thermophysical
response of materials/substances is differential scanning calorimetry (DSC)
[2], and it is widely used to perform this kind of characterization of PCM at
laboratory scale. This technique consists on heating up one sample and one
blank (which is a completely empty crucible) in order to remove the signal
from the crucible by itself. The obtained signal will be the thermophysical
behavior of the sample under study. Both crucibles must be heated under an
inert atmosphere (normally 50-80 ml/min N2 flow) by using a constant and
controlled heating rate. For PCM analysis slow heating rate is recommended
(~0.5 C/min) [15].
2.1. Thermophysical Characterization Performed with DSC
Barreneche et al. [16] studied the thermophysical behavior of d-mannitol

when it is cycled in differential scanning calorimeter. D-mannitol used for this
study was the one commercialized by QUIMIVITA and DSC equipment used
was DSC-822e by Mettler Toledo. A total of 14 samples were analyzed, three
were cycled once, three were cycled twice, five were cycled three times, and
three samples were cycled five times applying DSC analysis. It was performed
a 0.5 C/min slow dynamic mode under 80 ml/min N2 flow. The crucibles used
were 40 l aluminum crucibles and 15 mg approx. was the sample size.
Thereby, different thermal behaviors were obtained beyond the samples
and cycles performed and in are represented Figure 7. The first sample
(sample 1) shows a single peak at 167 C corresponding to 247 kJ/kg (Figure
7.a), the second thermal behavior (Sample 2 - Figure 7.b) has double peak at
156 C and 167 C related with 238 kJ/kg, and the third thermal behavior
(Sample 3 - Figure 7.c) is a single peak at 157 C linked with 234 kJ/kg. All
curves represented in Figure 7 were the result of the third thermal cycle on
DSC experimentation. DSC-822e equipment has 0.1 C and 3 kJ/kg of
inaccuracy.
2.a) Sample 1 phase: 246 Jg-1 (167 C)
a
2.b) Sample 2 transition and phases:

238 Jg-1 (157 C and 167 C)
2.c) Sample 3 phase: 243 Jg-1 (157 C)
Figure 7. Three different thermal behaviors obtained with DSC analysis of d-mannitol
during melting process.
In addition it was found that polymorphic transformations presented by d-

mannitol change the melting point of the substance while enthalpy is almost
constant after 5 cycles. In most cases, PCM application depends on phase
change temperature and it is a key parameter to take into account during the
material selection step.
Furthermore, the cooling performance of d-mannitol was also studied by
Barreneche et al. [16] and the phase change is regular and besides, subcooling
behavior is observed being the solidification temperature around 119 C under
same experimental conditions.
2.2. Morphological Characterization
Polymorphic analysis of d-mannitol was performed in order to associate

the thermal behavior obtained by DSC analysis with a specific crystalline
phase.
Figure 8. On the left FTIR bands of d-mannitol with no cycles of DSC; on the right
FTIR bands of d-mannitol cycled 5 times by DSC.
Table 3. Comparison of FTIR characteristic bands of d-mannitol with the

selected bands obtained
Characteristic bands (cm-1) [14] 1210 1081 1019 959 930

-phase
Selected bands (cm-1) 1209 1081 1018 959 929
Characteristic bands (cm-1) [14] 1193 1088 1025 968 932

-phase
Selected bands (cm-1) 1193 1088 1020 967 930
These analyses were performed by FT-IR and the equipment used is a

Thermo IZ10 and it was used coupled to an attenuated total reflectance
accessory (ATR) of diamond. Accordingly, the bands obtained with FT-IR
spectrograms were compared between 2000 and 500 cm-1 wave length with
those obtained by Burger et al. [14]. Two different samples were analyzed; the
first one is a new pure sample of d-mannitol and the second one was a sample
cycled 5 times with DSC.
FT-IR results are shown in Figure 8 and those demonstrate that there are
some changes in the structure of d-mannitol.
Moreover, the FT-IR bands selected by Burger et al. [14] to characterize
each d-mannitol structure are listed in Table 3 and those phases are compared
with the bands obtained as results by FT-IR spectroscopy.
Table 3 shows that the characteristic FT-IR bands correspond to -phase
in the case of pure new sample of d-mannitol and -phase for sample which
was cycled 5 times in DSC. This morphological structure can be correlated
with the first and the third thermal behavior shown in Figure 7.a and Figure
7.c, respectively.
2.3. Analysis of Occurrences
Based on DSC analysis, the percentage of occurrences of each thermal

behavior obtained depending on how many times the sample has been cycled
were calculated and results are shown in Table 4.
When the sample was analyzed once, all the samples solidified as -phase.
After the second cycle, the metastable -phase formation started. When the
samples were cycled three times, 50% presented a double peak (transition
between phases) and 50% solidified as -phase. Then, all samples showed a
double peak during the fourth cycle. Finally, the -phase is formed again
33.3% of the time because this phase is more stable than the other one and its
formation is favored.
Table 4. Occurrence percentage where the d-mannitol has a single peak

or double peak performance
1 Peak 2 Peaks
Tm, (C) Tm, (C) Tm, (C) Tm, (C)
0% 100% 0%
1st Cycle
--- 168.5 1 --- ---
18% 18% 64%
2nd Cycle
157 0 166 0 152 3 167 2
50% 0% 50%
3rd Cycle
156 0 --- 153 2 167 1
0% 0% 100%
4th Cycle
--- --- 152 2 164 0
0% 33.33% 66.66%
5th Cycle
--- 164 0 149 1 164 0
Several working temperature ranges were studied to establish the working

range to be considered when d-mannitol is used as PCM for TES. This
working range has to include all phases formed under the studied conditions
and the enthalpy of fusion has to be almost constant within this temperature
range. For all these reasons, the working temperature range recommended to
use when d-mannitol is selected as PCM was between 135 C and 175 C.
3. THERMAL BEHAVIOR OF D-MANNITOL WHEN THIS

MATERIAL IS USED AS PCM AT PILOT PLANT SCALE
Researchers from GREA research group (University of Lleida, Spain)
designed and built a high temperature pilot plant in order to test several
materials to be used as PCM in different applications. One study was
performed using d-mannitol as PCM and this substance was tested at pilot
plant scale by Gil et al. [17]. Additionally, results obtained were compared
with those obtained at laboratory scale.
The high temperature pilot plant (Figure 9) consist on an electrical boiler
of 24 kWe to heat up the heat transfer fluid (HTF) acting as energy source
during the charging process and an air heat exchanger of 20 kWe to cool down
the HTF and simulate the real energy consumption.
Figure 9. High temperature pilot plant to test PCM for solar cooling applications.
D-mannitol was located inside a shell-and tubes heat exchanger type

storage tank (Figure 10.a). The PCM was located inside of the shell and the
HTF circulated inside the 49 square pitch distributed tubes bundle. The tubes
were bended in U shape and they were connected in each side of the collectors
which distributes the HTF.
Temperature is controlled with fifteen temperature sensors which are
located as Figure 10.b shows. All temperature sensors were Pt-100 and were
calibrated before their installation in the storage tank with an accuracy of 2%.
Two experiments were performed to evaluate the thermal properties of d-
mannitol as PCM at pilot plant scale. The experiments performed consisted on
PCM solidification at different cooling rates. Temperature conditions details of
both experiments are listed in Table 5.
Figure 10. TES unit constructed: a) U shape tubes bundle; b) position of the
temperature sensors.
Initially, PCM was solidified applying the appropriated cooling rate

established in each experiment. Then, the PCM was melted again in order to
measure the melting temperature range and the melting enthalpy. In both
experiments, the HTF flow rate used was 3 m3/h. The initial PCM temperature
and HTF temperature were the same. Figure 11 shows the results of the PCM
melting process during the experiment A. In this experiment, the obtained
melting temperature range was between 160170 C and that range
corresponds to d-mannitol -phase temperature range, this phase is a stable
crystalline phase. The melting enthalpy of this experiment calculated was
149.0 kJ/kg.
Table 5. Characteristics of the experiments performed at pilot plant scale
Experiment Initial PCM Final PCM Temperature Cooling rate

temperature temperature gradient [C] [C/min]
[C] (solidification) [C]
A 187 145 42 0.17
B 200 130 70 0.36
210
200
190
Temperature [C]
180
170
160
150
140
PCM temperature
130
HTF inlet temperature
120
0:00 0:28 0:57 1:26 1:55
Time [h:min]
Figure 11. D-mannitol melting process in the pilot plant during Experiment A.
210
200
190
Temperature [C]
180
170
160
150
140
PCM temperature
130
HTF inlet temperature
120
0:00 0:28 0:57 1:26 1:55
Time [h:min]
Figure 12. D-mannitol melting process in the pilot plant during Experiment B.
Figure 13. XRD analysis of d-mannitol cycled in the TES unit (-phase - sample from
Experiment A).
Figure 14. XRD analysis of d-mannitol cycled with TES unit (-phase - sample from
Experiment B.
Moreover, results obtained for experiment B are plotted in Figure 12. The
temperature range of phase change obtained was between 150 C and 162 C.
This temperature range corresponds to a-phase or d-phase as Table 2 listed.
In order to discern which phase is obtained after each experiment
performed, two samples were analyzed X-ray diffraction (XRD). Each sample
was gathered after experiment A and experiment B, respectively.
Diffractograms of these two samples analyzed are presented in Figure 13 and
Figure 14.
These results were compared with patterns of XPERT HIGHSCORE
PLUS (Panalyatical) database. The sample gathered after experiment A
corresponds to -phase (Figure 13) and the sample gathered after experiment
B is linked to -phase (Figure 14).
Samples gathered after each experiment (A and B) were also analyzed
with DSC in order to compare the results obtained at pilot plant scale with
those obtained at laboratory scale considering exactly the same crystalline
phase.
The DSC results and pilot plant results are overlapped in Figure 15 where
curves of melting enthalpy vs. temperature are plotted. The melting range
obtained by DSC is shorter than the high temperature pilot plant. This fact is
due to the amount of material and the dimension of the equipment used.
Figure 15. Comparison of results obtained in the TES unit and obtained by DSC.
4. COMPARISON OF D-MANNITOL AND HYDROQUINONE

WHEN USED AS PCM FOR TES
AT PILOT PLANT SCALE
Literature and DSC research of many phase change materials (PCM)

candidates for solar cooling applications and selection were studied by Gil et
al. [18]. Hydroquinone and d-mannitol were the PCM selected to be tested and
compared at pilot plant scale. Different experiments with different flow rates
and HTF temperatures were performed to analyze the viability of the use of
these materials in thermal energy storage systems for solar cooling
applications.
These two materials (d-mannitol and hydroquinone) were tested in the
high temperature pilot plant described above.
For both PCM, regular thermal behavior was obtained beyond the
experiments performed in the pilot plant and even though hydroquinone
presented subcooling in the DSC, it did almost not appear in pilot plant scale,
however, when d-mannitol was used big subcooling was detected during the
discharging process.
Furthermore, the effective heat transfer coefficient between the storage
material and the heat transfer fluid (HTF) was calculated: the effective heat
transfer [18] was 0.86 for hydroquinone, while it was 0.88 for d-mannitol.
Therefore, almost all the heat that is given by the HTF during the charging
process was recovered when the discharging occurs. For the same boundary
conditions, the energy stored by d-mannitol was higher than that for
hydroquinone; in particular, the enhancement was about 30% and 20% during
the charging and the discharging processes even though the enhancement of
the latent heat was only 10% and 16%, respectively. Thereby, these
experiments show that d-mannitol has higher energy storage capacity but it
showed subcooling every time was tested.
CONCLUSION
Thermal energy storage (TES) systems are proposed as one of the most
powerful technologies to facilitate a greater use of renewable energy by
storing the energy produced so it can coincide with demand. TES systems are
able to store energy as latent heat storing energy using the latent heat produced
when a phase change state occur using phase change materials (PCM). PCM
candidates must fulfil several conditions to be used as storage materials:

melting point of PCM must be closed to selected work temperature range; it
must own high specific heat in order to provide sensible heat and high thermal
conductivity (solid and liquid state) to support charging and discharging
processes inside the storage system. D-mannitol has a phase change
temperature at 167 C and its phase change enthalpy is around 316 kJ/kg.
These thermophysical properties turn d-mannitol as a perfect candidate to be
used as PCM in solar cooling applications and it was studied with this purpose.
In this chapter, thermophysical properties of d-mannitol were
characterized performing differential scanning calorimetry under dynamic
mode using a 0.5 K/min heating rate between 25 C and 200 C and its
polymorphic transformation was studied with FT-IR and two different phases
were identified: -phase and -phase. Then, a range temperature was
established to work with this substance as phase change material: this working
temperature is between 135 C and 175 C. This working range includes the
phase change transformation of the two morphological phases under analysis
( and ).
To test the d-mannitol thermal behaviour at pilot plant scale 150 kg of d-
mannitol were introduced in a storage tank which was design as shell-and-
tubes heat exchanger. Two experiments were performed to evaluate the
thermal properties of d-mannitol as PCM at pilot plant scale and to confirm the
results of a previous study at laboratory scale. It was concluded that applying
different cooling conditions produces d-mannitol polymorphic changes.
Moreover, it was shown that the working range (between 135C and 175C)
defined in the first study is adequate for pilot plant experiments because the
thermal results obtained in the pilot plant are inside this working range.
REFERENCES
[1] Abhat. Low temperature latent heat thermal energy storage: heat storage
materials. Solar Energy 30,313332: 1983.
[2] Mehling H, Cabeza LF. Heat and cold storage with PCM. Springer-
Verlag; 2008. ISBN-13: 9783540685562.
[3] L.F. Cabeza, A. Castell, C. Barreneche, A. de Gracia, A.I. Fernndez.
Materials used as PCM in thermal energy storage in buildings: A review.
Renewable and Sustainable Energy Reviews 15, 1675-1695: 2011.
[4] R. Baetens, B.P. Jelle, A. Gustavsen. Phase change materials for

building applications: A state-of-the-art review. Energy & Buildings 42,
1361-1368: 2010.
[5] S.D. Sharma, K. Sagara. Latent heat storage materials and systems: A
review. International Journal of Green Energy 2(1), 1-56: 2005.
[6] M.M. Farid, A.M. Khudhair, S.A.K. Razack, S. Al-Hallaj. A review on
phase change energy storage: materials and applications. Energy
Conversion and Management 45, 15971615: 2004.
[7] Zalba, J.M. Marn, L.F. Cabeza, H. Mehling. Review on thermal energy
storage with phase change: materials, heat transfer analysis and
applications. Applied Thermal Engineering 23, 251283: 2003.
[8] E. Gnther, S. Hiebler, H. Mehling. Determination of the heat storage
capacity of PCM and PCM-objects as a function of temperature. Proc.
ECOSTOCK, 10th Int. Conference on Thermal Energy Storage
(Stockton, USA). 2006.
[9] J. Cot-Gores, A. Castell, L.F. Cabeza. Thermochemical energy storage
and conversion: A-state-of-the-art review of the experimental research
under practical conditions. Renewable and Sustainable Energy Reviews
16(7), 5207-5224: 2012.
[10] K. E. NTsoukpoe, H. Liu, N. Le Pierres, L. Luo. A review on long-term
sorption solar energy storage. Renewable and Sustainable Energy
Reviews 13, 2385-2396: 2009.
[11] Gil, M. Medrano, I. Martorell, A. Lzaro, P. Dolado, B. Zalba. State of
the art on high temperature thermal energy storage for power generation.
Part 1 concepts, materials and modellization. Renewable and
Sustainable Energy Reviews 14: 31-55: 2010.
[12] M. Medrano, A. Gil, I. Martorell, X. Potau, L.F. Cabeza. State of the art
on high temperature thermal energy storage for power generation. Part 2
case studies. Renewable and Sustainable Energy Reviews 14, 56-72:
2010.
[13] L.A. Chidambaram, A.S. Ramana, G. Kamaraj, R. Velraj. Review of
solar cooling methods and thermal storage options. Renewable and
Sustainable Energy Reviews 15, 3220-3228: 2011.
[14] Burger, J.O. Henck, S. Hetz, J.M.Rollinger, A.A.Weissnicht, H. Stttner.
Energy/Temperature Diagram and Compression behavior of the
Polymorphs of D-Mannitol. Journal of Pharmaceutical Science 89, 457-
468: 2000.
[15] Barreneche, A. Sol, L. Mir, I. Martorella, A.I. Fernndez, L.F.
Cabeza. Study on differential scanning calorimetry analysis with two
operation modes and organic and inorganic phase change material

(PCM). Thermochimica Acta 553, 23-26: 2013.
[16] Barreneche, A. Gil, F. Sheth, A.I. Fernndez, L.F. Cabeza. Effect of d-
mannitol polymorphism in its thermal energy storage capacity when it is
used as PCM. Solar Energy 2013, http://dx.doi.org/10.1016/
j.solener.2013.05.023.
[17] Gil, C. Barreneche, P. Moreno, C. Sol, A.I. Fernndez, L.F. Cabeza.
Thermal behaviour of D-mannitol when used as PCM: Comparison of
results obtained by DSC and in a thermal energy storage unit at pilot
plant scale. Applied Energy 2013, http://dx.doi.org/10.1016/
j.apenergy.2013.04.081.
[18] Gil, E. Or, G. Peir, S. lvarez, L.F. Cabeza. Material selection and
testing for thermal energy storage in solar cooling. Renewable Energy
57, 366-371: 2013.
In: Mannitol ISBN: 978-1-62808-762-8
Chapter 5
CHIRAL PHOSPHOROUS LIGANDS DERIVED

FROM D-MANNITOL:
SYNTHESIS AND THEIR APPLICATIONS
IN ASYMMETRIC CATALYSIS
Yingwei Zhao, Lei Yang and Hanmin Huang

State Key Laboratory for Oxo Synthesis and Selective Oxidation,
Lanzhou Institute of Chemical Physics, Chinese Academy of Sciences,
Lanzhou, China
ABSTRACT
As a kind of readily available carbohydrate, D-mannitol has been
widely used in the synthesis of chiral phosphorous ligands. This chapter
reviews the design and synthesis of various mono- and diphosphorous
ligands based on D-mannitol backbone. Also, the successful applications
of these ligands in asymmetric catalysis, such as enantioselective
hydrogenation and enantioselective conjugate addition, are summarized.
Fax: (+86)931-496-8129 E-mail: hmhuang@licp.cas.cn.

88 Yingwei Zhao, Lei Yang and Hanmin Huang
1. INTRODUCTION
In the past twenty years, the design and synthesis of highly efficient chiral
ligands for asymmetric catalysis had made considerable achievements. [1]
Among the development of suitable ligands for specific transition metal
catalyzed enantioselective reactions such as asymmetric hydrogenations, [2, 3]
much effort has been made to construct chiral backbones by utilizing the
resources existing in natural chiral pools. Carbohydrates have been widely
used for enlarging the amounts of potential superior ligands because they are
easily accessible, commercially available, and often of high enantiomeric
purity. [4] Most importantly, their highly functionalized property makes it
straightforward to design novel ligands bearing specific structures through
modifications.
D-Mannitol is commonly obtained via the hydrogenation of fructose,
which is formed from either starch or sucrose. As an important carbohydrate, it
has been extensively used in organic synthesis with several applications, such
as ligands, [5] polymers and the preparation of small chiral building blocks,
[6] and key intermediates in total synthesis. [7] Versatile ligands using D-
mannitol as a backbone have been synthesized, and successfully applied in
asymmetric catalytic reactions. The role of functional group derived from
mannitol in the ligands structure could be divided into two types: (1) as the
main chiral backbone; and (2) as an additional group. Genarally, the mannitol
fragment could provide fair degree of rigidity and flexibility to enhance the
enantioselectivity of the whole ligand. In some case, the mannitol moiety may
also act as hemilabile coordination group. [8, 9]
2. METHODS FOR MANNITOL MODIFICATION

In order to introduce the mannitol backbone into chiral ligands efficiently,
the transformation of available D-mannitol into a functional chiral diol contain
a C2 axis through chemical modification is preferentially considered. In fact,
several methods for such transformation had been well developed Generally,
the first step is the acetalization of D-mannitol with an aldehyde, or
ketalization with a ketone.
The condensation of D-mannitol with benzaldehyde in the presence of
concentrated sulfuric acid led to 1,3:4,6-Di-O-benzylidene-D-mannitol 1
(Scheme 1) [10, 11]. The dehydration occurred between the two hydroxyl
Chiral Phosphorous Ligands Derived from D-Mannitol 89
groups at C1 and C3 positions and other two hydroxyl groups remained with
furnishingthe chiral 1,4-diol.
Scheme 1. Condensation of D-mannitol with benzaldehyde.
Scheme 2. Synthesis of the chiral 1,2-diol.
The reaction of D-mannitol with ketones is of somewhat complex. The

products distribution depends on the reaction conditions. In the presence of
SnCl4, D-mannitol could be facile selectively ketalized by two equivalents of
2,2-dimethoxypropane to afford chiral 1,2-diol 2 (Scheme 2) [12].
Scheme 3. Synthesis of 1,4-disubstituted 1,4-diol from D-mannitol derived 1,2-diol 2.

The 1,2-diol 2 could be further applied in constructingother chiral

backbones.
The related procedure included deoxygenation of 2 into the corresponding
olefine 3 through the method discovered by Corey [13], hydrogenation,
deprotection of 4 by acid hydrolysis to afford 3,4-dideoxy-D-threo-hexitol 5
which is a common chiral synthon, and subsequently the selective benzylation
of the primary hydroxy groups to afford 1,6-di-O-benzyl ether 6. [14, 15]
Scheme 4. Transformation of 1,2-diol 2 into benzyl protected 1,4-diol 8.
Another approach for 1,4-diol backbone synthesis from ketalized mannitol

2 concerning the prior O-Benzylation of the remaining alcoholic groups and
acidic cleavage of the acetal protective groups to yield the tetrol 7. [16]
Both primary hydroxymethyl groups in turn were converted to methyl
groups via tosylation and subsequent reduction with LiAlH4(LAH) (8)
(Scheme 4).
Scheme 5. Synthesis of isopropylidene ketal protected 1,4-diol.

Similar 1,4-diol 10 was accomplished through a concise two steps

procedure. The selective tosylation of the tetrol 9 and subsequent reduction
were also included (Scheme 5). [17, 18]
Scheme 6. Synthesis of isopropylidene ketal protected 1,4-diols with specific

configuration.
Using the tetrol 9 as starting material, the chiral 1,4-diols like 10a with
specific spatial configuration could be obtained (Scheme 6). [19, 20]
Depending on the specific reaction conditions, two diastereomeric
bis(epoxides) (11a and 11b) were selectively prepared, respectively. The
intramolecular SN2 reaction of the tosylate reversed the absolute configuration
of C2 chiral center from R to S. The bis(epoxides) were then reduced with
LiAlH4 in THF to the corresponding diols (10a and 10b). The epoxides could
also serve as electrophiles for copper-mediated ring opening to afford 12a and
12b. This synthetic strategy allows flexibility in steric tuning of the final
phospholane ligands.
Scheme 7. Synthesis of D-isomannide.
The dehydration of D-mannitol itself could take place in the presence of

concentrated hydrochloride acid to afford 1,4:3,6-Dianhydro-D-mannitol 13
(also called D-isomannide) in satisfying yield (Scheme 7). [21] The
etherification of hydroxyl groups takes place at the C1 and C3 positions,
leading to a backbone containing cis fused tetrahydrofuran rings which have
relatively rigid conformation.
3. PHOSPHOROUS LIGANDS AND THEIR APPLICATIONS

3.1. 1,4:3,6-Dianhydro-D-Mannitol As Backbone
In 2004, Huang and coworkers firstly reported a method for introducing

1,4:3,6-dianhydro-D-mannitol backbone into BINOL derived
monophosphites [22]. A new series of chiral ligands 15 with double six-
membered-ring backbone abbreviated ManniPhos were developed. These
ligands can be conveniently synthesized in two steps starting from diol 1, as
illustrated in Scheme 8 [22].
Scheme 8. Synthesis of 1,4:3,6-dianhydro-D-mannitol derived ManniPhos.
Table 1. Asymmetric hydrogenation of N-acetylphenylethenamine

catalyzed by Rh-15
entry ligand ee (%) (config)

1 15a 99.8 (S)
2 15b 99.8 (S)
3 15c 99.4 (S)
4 15d 97.7 (S)
5 15e 98.1 (S)
6 15f 99.6 (S)
7 15g 99.5 (S)
8 15h 91.3 (R)
9 15i 92.1 (R)
10 15j 49.7 (R)
11 15k 9.7 (R)
Solvent: CH2Cl2; p(H2) = 10 atm; T = 20 C; reaction time: 12 h; substrate :
[Rh(COD)2]BF4: 15 = 1 : 0.01 : 0.022; 100% conversion was obtained in all cases.
These monophosphites are efficient ligands for asymmetric hydrogenation

of N-acetylphenylethenamine. However, the enantiomeric excess depends
strongly on the moiety of the P/O heterocycle in the chiral ligands. Values of
ee up to 99.8 % have been achieved with Rh-15a and Rh-15b as catalysts and
ligands 15j and 15k derived from conformationally flexible biphenols gave
only 9.7-49.7 %, demonstrating that R-BINOL is matched cooperatively to the
corresponding D-mannitol derived backbone. The enantioselectivities
produced by these ligands are also affected by the -OR groups contained in the
ligands, which can be attributed to their effects on the degree of rigidity and
flexibility of the D-mannitol derived double-ring backbone. Less bulky
substituents generally induce a higher ee value, and the simplest ligand 15a is
the most efficient. These ligands were also found to exhibit excellent catalytic
activities and enantioselectivities in Rh-catalyzed hydrogenation of dimethyl
itaconate, -dehydroamino acid esters, -(acylamino)acrylates and
-phthalimide acrylates [23, 24, 25].
Scheme 9. Synthesis of ionic ManniPhos.
To make the reutilization of these efficient ligands, further modification of

the chiral backbone was carried out through incorporation of an ionic tag to
the above chiral ligands, affording ionic ligands 16a-e (Scheme 9) [26]. All
the ionic catalysts showed high activities (100% conversions in 1 h) and
excellent enantioselectivities (99% or >99% ee) in asymmetric hydrogenation
of N-acetylphenylethenamines. The ligands could be recycled in an ionic
liquid media [bmim][BF4]: 95% conversion and 97% ee were still obtained in
the tenth run with 16d as chiral ligand.
3.2. 1,2:5,6-Dianhydro-D-Mannitol As Backbone
Scheme 10. Synthesis of DIMOP.
Chans group firstly prepared the ligand 1,2,5,6-di-isopropylidene-3, 4-

bis(diphenylphosphino)-D-mannitol [abbreviated as D-DIMOP (17)] through
the simple reaction of diol 2 with chlorodiphenyl phosphine in dry THF in the
presence of Et3N (Scheme 10). [27]
Scheme 11. Asymmetric hydrogenation catalyzed by DIMOP.
High enantioselectivities were found in the asymmetric hydrogenation of

2-acetamidoacrylic acid catalyzed by the cationic rhodium complex of 17
(Scheme 11). The bulky ketal groups on the chiral backbone were thought to
be significant in increasing the rigidity of the phosphinite ligands in their
transition-metal complexes.
The group then reported the preparation and application of several chiral
P,N-ligands (19-21) derived from 1,2:5,6-di-O-cyclohexylidene-D-mannitol
(18) which could be obtained by the method similar as the synthesis of diol 2
(Scheme 12). [28]
The ligands have the axially chiral binaphthyl moiety and the
phenylcarbamate substituents at either the 3- or 4-position of D-mannitol. The
corresponding Rh-complexes were applied as catalyst precursors in the
asymmetric hydroformylation of styrene.
However, only 19a gave notable results (22% conv., 89/11 b/n, and 50%
ee). Chiral diphosphite ligands 22a and 22b were also synthesized from diol
18 and corresponding phosphorochlorides (Scheme 13). [29] The ligand 22b

gave higher ee value (75%) in the asymmetric hydroformylation of styrene,
however, the conversion was low (2.4%).
Scheme 12. Synthesis of 1,2:5,6-dianhydro-D-mannitol derived phosphites.
Scheme 13. Synthesis of 1,2:5,6-dianhydro-D-mannitol derived diphosphites.
The following work carried out by Wangs group [30] refer to the
application of these phosphite ligands in the asymmetric Cu-catalyzed
enantioselective conjugate addition of diethylzinc to cyclic enones.
The value of the enantiomeric excess is controlled by the cooperative
effect between the mannitol backbone (C-3 and C-4) and the binaphthol
phosphite moiety.
When ligand 22a was used, up to 71% ee was obtained for addition
reaction of cyclopentenone under the optimized conditions.
Scheme 14. 1,2:5,6-dianhydro-D-mannitol derived phosphites with BIONL and

H8-BIONL moieties.
Table 2. Cu-catalyzed enantioselective 1,4-addition of ZnEt2 to

2-cyclohexenone
entry ligand ee (%) (config)

1 21a 32 (S) (-30 C)
2 21b 7.9 (R)
3 21c 7 (S)
4 21d 75 (R)
5 22a 53 (S)
6 22b 47 (R)
7 22c 45 (S)
8 22d 83 (R)
Reaction conditions: Cu(OTf)2 (0.005 mmol), ligand (0.01 mmol), ZnEt2 (1.0 M in
hexane, 0.6 mmol), 2-cyclohexenone (0.25 mmol), toluene (4 mL), 0 C, 4 h.
The authors then introduced H8-binaphthyl moiety into the D-mannitol

skeleton instead of traditional binaphthyl group, affording new ligands 22c,
22d and 23a-d (Scheme 14). They were then applied them in the Cu-catalyzed
asymmetric 1,4-conjugate addition of dialkylzincs to cyclic and acyclic enones
(Table 2). The results indicated that the enantioselectivities of the products
could be significant enhanced when R-H8-BINOL (d) was used as the axially
chiral substituent (75% and 83% ee). In contrast, the ligands containing S-H8-
BINOL (c) group afforded only low ee values. This result indicated that the
stereogenic centers of the D-mannitol skeleton and the axially chiral diaryl
moieties of ligands had a synergic effect on the enantioselectivity of the
reaction.
3.3. 1,4:3,6-Dianhydro-D-Mannite As Backbone
Scheme 15. Synthesis of 1,4:3,6-dianhydro-d-mannite derived phosphites.
The C2-symmetric 1,4:3,6-dianhydro-d-mannite 13 exhibits a vaultlike

geometry with two hydroxyl groups on the concave side. Reetzs group firstly
carried out the reactions of 13 with chlorophosphoric acid diaryl ester (2 equiv
each) (Scheme 15), providing an efficient protocol for the synthesis of many
different phosphites ligands 24 (Table 3). [31]
They were then applied these ligands in the Rh-catalyzed hydrogenation
of dimethyl itaconate (Table 3). The catalysts derived from ligands 24b and
24c which contain (S)- and (R)- binaphthol, respectively, in the P/O
heterocycle behave differently. The ee values of 88% and 95% attained prove
that the chirality in the P/O heterocycles is decisive and that the R-selective
combination 24/(R)-binaphthol is the matched case. In the case of the
unsubstituted biphenol derivative 24d, only 39% ee was obtained. In contrast,
ligand 24e with ortho methyl groups shows an entirely different behavior.
Interestingly, 24f with sterically demanding tert-butyl group leads to low
activity as well as poor enantioselectivity.
Table 3. Enantioselective hydrogenation catalyzed by Rh-24
entry (RO)2PCl ligand ee (%) (config)

1 24a 21.0 (S)
2 24b 87.8 (S)
3 24c 94.5 (R)
4 24d 38.9 (S)
5 24e 96.8 (R)

entry (RO)2PCl ligand ee (%) (config)

6 24f 5.2 (R)
7 24g 49.3 (S)
Reaction conditions: substrate: catalyst (s:c) = 1000 : 1; t = 20 h; T = 20 C; ligand :

rhodium = 1:1.
Scheme 16. 1,4:3,6-dianhydro-d-mannite derived monophosphites.
Surprisingly, subsequent investigates indicated that the monophosphite

units 25a and 25b (Scheme 16) also gave high stereoselectivities in the
hydrogenation of dimethyl itaconate (97.8% and 95.2% ee were obtained in
the presenceof 0.1 mol% of catalyst, respectively). [32]
This could be attribute to the fact that one of the three ether moieties
contained in the ligand serves as a hemilabile ligand and thus reduces the
conformational freedom.
3.4. Phosphorous Ligands from D-Mannitol Derived 1, 2, 5, 6-

Tetrol
3.4.1. 2,2'-Bipyran-3,3'-Diol as Backbone

Dings group synthesized a novel C2-symmetric bisphosphinite ligand 26
containing two tetrahydropyran rings, starting from the bis(epoxides) 11a
which could be synthesized from the tetrol 9 (Scheme 17). [33]
Scheme 17. Preparation of C2-symmetric bisphosphinite ligand 26.
Scheme 18. Asymmetric allylation of 7 with dimethyl malonate promoted by Pd-26

complex.
To evaluate the asymmetric induction efficiency of 26, palladium-

catalyzed allylic substitution of 1,3-diphenyl-2-propen-1-yl acetate with
dimethyl malonate was examined (Scheme 18). Under the optimized
conditions, the allylation product could be obtained in up to 91.2% ee.
3.4.2. RoPhos-Type Ligands

In 1998, Brners group firstly developed a new family of phosphine
ligands named RoPhos derived from D-mannitol. [16] These ligands could
be regarded as ether-functionalized DuPhos. The RoPhos matched high
efficiency of DuPhos-type ligands in catalytic asymmetric hydrogenation, but
the synthetic procedure was quite simpler than that of DuPhos (Scheme 19).
Scheme 19. Synthesis of RoPhoses.
Starting from D-mannitol, the diol 8 could be obtained by the protocol

described in Scheme 4. Esterification of the hydroxy groups with thionyl
chloride and in situ oxidation of the intermediate sulfite with catalytic amounts
of RuO4 gave the cyclic sulfate 27. The intermediate 27 could be used as a
platform molecule for synthesis of ligand 29a by simply changed the O-
protective groups.
The sulfates were individually reacted with 1,2-diphosphinoethane or 1,2-
diphosphinobenzene in the presence of n-BuLi to give bisphospholanes 28a,
29a, and 30a.
Table 4. Asymmetric hydrogenation with Rh complexes of RoPhos type
entry ligand time for 50% ee (%) (config)

conversion (min)
1 28a 28 98.0 (R)
2 29a 9 98.9 (R)
3 30a 8 99.1 (R)
Condition of the hydrogenation: 1.0 atm overall pressure over the solution. The
experiments were carried out under standard conditions with 0.01 mmol of
precatalyst and 1.0 mmol of prochiral olefin in 15 mL of solvent.
Scheme 20. Synthesis of RoPhos type hydroxyl phospholane ligands.
The complexes of RoPhos with Rh(I) were tested in the asymmetric

hydrogenation of prochiral functionalized olefins (Table 4). In all cases, high
enantioselectivities were achieved. Although the catalytic differences between
ethylene- and phenylene-bridged phospholanes as well as the influence of the
different O-protective groups upon the intrinsic of the 2,4-

dimethylphospholane complex are small, the effect is significant. Thus, by the
proper choice of these structural features, fine-tuning of the catalyst toward the
special requirements of each substrate is possible.
Soon after the discovery of RoPhos, Browns group synthesized a similar
ligand containing a phosphineborane moiety and a phospholane ring. [34]
Zhang and coworkers designed and synthesized a similar type of chiral
diphosphine ligands 36a, 37a and 38a from D-mannitol with free hydroxyl
groups (Scheme 20). [17, 18 35] In order to efficiently remove the O-
protecting group in the presence of phosphine, isopropylidene ketal protected
mannitol derivatives 10a and 12a were selected as starting materials instead of
benzyl protected 8.
The hydroxyl monophospholane 34a was used as catalyst for Baylis-
Hillman reaction, but only 17% enantioselectivity was obtained.
Table 5. Rhodium catalyzed asymmetric hydrogenation of

dehydroamino acid
entry R ligand ee (%)

1 H 37a >99
2 CH3 37a 98
3 H 38a >99
4 CH3 38a >99
The reaction was carried out at rt under 3 atm of H 2 for 9 h. The reaction went with
100% conversion.
Then the hydrogenation of -dehydroamino acid was chosen as a model

reaction for examination the catalytic properties of these chiral diphosphine
ligands (Table 5). Surprisingly, the isopropylidene-protected phospholane 35a
does not work in the hydrogenation reaction for this substrate. However, Rh
complex with the corresponding hydroxyl phospholane 37a is an excellent
hydrogenation catalyst. The more sterically hindered Et-phospholane 38a
provided slightly higher enantiomeric excess compared with Me-phospholane
37a. The enantioselectivities obtained with these hydroxyl phospholanes are
comparable to the results achieved with Rh-DuPhos catalysts and higher than
that of with an Rh-RoPhos catalyst.
Zhangs group also synthesized a ferrocene-bridged polysubstituted
phospholane ligand 39 with the same phosphorous cycle as ligands 35a and
36a. The Rh-complex with this ligand showed high enantioselectivity and
reactivity in the asymmetric hydrogenation of -dehydroamino acid. Up to
over 99% ee and 10 000 TON were achieved with this catalytic system.
Riegers group developed a flexible approach to generate monodentate
phosphoramidites differing in the absolute configuration of carbon atoms and
the steric demand of the substituents. Refluxing the isopropylidene ketal
protected diols with hexamethyltriaminophosphane (HMTAP) in toluene could
afford phosphoramidites 43-47 (Scheme 21). [19, 20] The synthesized Rh
complexes bearing two monodentate phosphoramidite ligands (43-47) were
tested in the asymmetric hydrogenation of -(acetamido)cinnamic acid. The
ligands 43a-47a and 43b only produced a small excess of N-acetyl-(R)-
phenylalanine with lower ee values ranging from 3 to 36% Ligand 47b
remarkably gave an 89% ee for (S)-enantiomer, which may be due to front
orientation effect of the phenyl fragments.
Scheme 21. Synthetic route to monodentate phosphoramidites.

Scheme 22. Synthesis of mono- and diphospholane Ligands.
Almost at the same time of Brner and Zhangs work, [36] RajanBabus
group [37, 38] also discovered a versatile route for the synthesis of various
functionalized mono and bisphospholane ligands. The RoPhos-like ligands
35b was also efficiently prepared with different absolute configuration. The
method also allowed the approach toward biphosphinite 48b and biphosphine
49b from the diol 10b. Ligand 50b bearing a potentially hemilabile tert-
butylthioether functionality at the -position was also successfully prepared
with the same synthetic strategy (Scheme 22).
These ligands were evaluated by palladium(0)-catalyzed addition of
dimethyl malonate to (E)-1,3-diphenylprop-2-enyl acetate (Table 6).
It is illustrated that the sense of induction is controlled by the chirality of
the C2 and C5 atoms in all cases. Excellent results could be obtained by using
phospholanes 33b and 35b. The appropriate phosphine 49b gave higher ee
than its diastereotopic structure 49a. The ligand 50 containing chelating
phosphorus and sulfur atoms did not lead to decent enantioselectivity.
Brners group introduced a couple of hydroxylmethyl groups into the
phospholane cycles for enhancing the solubility of the corresponding Rh
complex 52 (Rh(BASPHOS)) in water [15]. Starting from the diol 6, the chiral
ligand 51 was successfully prepared in good yield (Scheme 23). The Rh(I)
complex 52 was applied in the asymmetric hydrogenation of the water-soluble
2-acetamido acrylic acid and its ester with water as solvent. Up to 99.6% ee
was obtained for the hydrogenation of the acid [15].
Table 6. Enantioselectivity in Pd-catalyzed allylations of dimethyl

malonate: effect of substituents on phospholane
entry ligand solvent L/Pd yield (%) ee (%) (config)

1 35a THF 1.6 99 >99 (R)
2 35b THF 1.6 99 94 (S)
3 33a toluene 2 98 93 (S)
4 33b toluene 2 99 94 (R)
5 49a CH2Cl2 2 98 ~0
6 49b CH2Cl2 2 92 63 (S)
7 50a CH2Cl2 1.1 98 60 (R)
8 50b CH2Cl2 1.1 99 44 (S)
Scheme 23. Synthesis of the water-soluble chiral tetrahydroxy diphosphine Rh(I)

catalyst.
RajanBabus group found that the hydroxyl RoPhoses 37a, 38a and 37b
could be successful used in the asymmetric hydrogenation of methyl
acetamidoacrylate in aqueous media. They could be recycled with no loss of
activity or selectivity in 1:1 methanol/water.
Scheme 24. Synthesis of neutral polyhydroxy phospholane derivative.
The authors also synthesized the free hydroxyl ligand 53, in which the
method for protection and de-protection of hydroxyls was different from that
described by Brners group (Scheme 24) [39]. Ligand 53 was found giving
the best results in hydrogenation in neat water. The enantioselectivity and
recyclability is outstanding with the catalyst derived from this ligand. In four
sequential runs, ~99% ee and >90% isolated yield (100% conversion) were
obtained for the hydrogenation of methyl acetamidoacrylate in neat water.
CONCLUSION AND PERSPECTIVE

Due to their highly functionalised and easily obtained features, the chiral
backbones derived from D-mannitol have found broad applications in
designing and synthesis of various types of chiral phosphorous ligands. These
ligands could effectively transfer chirality in many asymmetric reactions,
mostly because of their rational balance ability of rigidity and flexibility in

attaching additional groups and potential hemilabile effect. The RoPhos and
ManniPhos are regarded as the most successful ligands among them. They are
readily synthesized and exhibit high activities and enantioselectivities in many
asymmetric reactions, typically hydrogenation. In addition, they can also be
easy modified to hydrophilic derivatives, making the recycle of catalysts
possible. Considering its easy functionalization and low cost, it is believed that
in conjugation with the vast development of transition metal catalysis, D-
mannitol derived ligands will be a powerful tool for the steric control on many
newly discovered reactions.
REFERENCES
[1] Kagan, H. B.; Fiaud, J. C.; Hoornaert, C.; Meyer, D.; Poulin, J. C.
Bulletin des Socits Chimiques Belges 1979, 88 (11), 923-931.
[2] Xie, J.-H.; Zhu, S.-F.; Zhou, Q.-L. Chem. Rev. 2010, 111 (3), 1713-
1760.
[3] Wang, D.-S.; Chen, Q.-A.; Lu, S.-M.; Zhou, Y.-G. Chem. Rev. 2011,
112 (4), 2557-2590.
[4] Steinborn, D.; Junicke, H. Chem. Rev. 2000, 100 (12), 4283-4318.
[5] Aravind, A.; Mohanty, S. K.; Pratap, T. V.; Baskaran, S. Tetrahedron
Lett. 2005, 46 (17), 2965-2968.
[6] Aravind, A.; Kumar, P. S.; Sankar, M. G.; Baskaran, S. Eur. J. Org.
Chem. 2011, 6980-6988.
[7] de Oliveira, P. S. M.; Ferreira, V. F.; de Souza, M. V. N. Quimica Nova
2009, 32 (2), 441-452.
[8] Nomura, N.; Jin, J.; Park, H.; RajanBabu, T. V. J. Am. Chem. Soc. 1998,
120 (2), 459-460.
[9] Bader, A.; Lindner, E. Coord. Chem. Rev. 1991, 108 (1), 27-110.
[10] Baggett, N.; Stribblehill, P. J. Chem. Soc. -Perkin Trans. 1 1977, 1123-
1126.
[11] Jiang, B.; Huang, Z. G.; Cheng, K. J. Heterocycles 2004, 63 (12), 2797-
2803.
[12] Schmid, C. R.; Bryant, J. D.; Dowlatzedah, M.; Phillips, J. L.; Prather,
D. E.; Schantz, R. D.; Sear, N. L.; Vianco, C. S. J. Org. Chem. 1991, 56
(12), 4056-4058.
[13] Corey, E. J.; Hopkins, B. Tetrahedron Lett. 1982, 23 (19), 1979-1982.
[14] Machinaga, N.; Kibayashi, C. J. Org. Chem. 1992, 57 (19), 5178-5189.
[15] Holz, J.; Heller, D.; Strmer, R.; Brner, A. Tetrahedron Lett. 1999, 40
(39), 7059-7062.
[16] Holz, J.; Quirmbach, M.; Schmidt, U.; Heller, D.; Strmer, R.; Brner,
A. J. Org. Chem. 1998, 63 (22), 8031-8034.
[17] Li, W.; Zhang, Z.; Xiao, D.; Zhang, X. J. Org. Chem. 2000, 65 (11),
3489-3496.
[18] Liu, D.; Li, W.; Zhang, X. Org. Lett. 2002, 4 (25), 4471-4474.
[19] Bayer, A.; Murszat, P.; Thewalt, U.; Rieger, B. Eur. J. Inorg. Chem.
2002, 2614-2624.
[20] Bayer, A.; Thewalt, U.; Rieger, B. Eur. J. Inorg. Chem. 2002, 199-203.
[21] Marr, A.; Wardell, J.; Cox, P. J Chem Crystallogr 1997, 27 (3), 161-166.
[22] Huang, H.; Zheng, Z.; Luo, H.; Bai, C.; Hu, X.; Chen, H. J. Org. Chem.
2004, 69(7), 2355-2361.
[23] Huang, H.; Liu, X.; Chen, S.; Chen, H.; Zheng, Z. Tetrahedron:
Asymmetry 2004, 15 (13), 2011-2019.
[24] Huang, H.; Liu, X.; Chen, H.; Zheng, Z. Tetrahedron: Asymmetry 2005,
16 (3), 693-697.
[25] Huang, H.; Liu, X.; Deng, J.; Qiu, M.; Zheng, Z. Org. Lett. 2006, 8(15),
3359-3362.
[26] Zhao, Y.; Huang, H.; Shao, J.; Xia, C. Tetrahedron: Asymmetry 2011, 22
(7), 769-774.
[27] Chen, Y.; Li, X.; Tong, S.-k.; Choi, M. C. K.; Chan, A. S. C.
Tetrahedron Lett. 1999, 40 (5), 957-960.
[28] Wang, L.-L.; Guo, R.-W.; Li, Y.-M.; Chan, A. S. C. Tetrahedron:
Asymmetry 2005, 16 (19), 3198-3204.
[29] Zhao, Q.-L.; Wang, L.-L.; Kwong, F. Y.; Chan, A. S. C. Tetrahedron:
Asymmetry 2007, 18 (16), 1899-1905.
[30] Zhao, Q.-L.; Wang, L.-L.; Xing, A.-P. Tetrahedron: Asymmetry 2010,
21 (24), 2993-2998.
[31] Reetz, M. T.; Neugebauer, T. Angew. Chem. Int. Ed. 1999, 38 (1-2),
179-181.
[32] Reetz, M. T.; Mehler, G. Angew. Chem. Int. Ed. 2000, 39 (21), 3889-
3890.
[33] Zhang, R.; Yu, L.; Xu, L.; Wang, Z.; Ding, K. Tetrahedron Lett. 2001,
42 (43), 7659-7662.
[34] Carmichael, D.; Doucet, H.; M. Brown, J. Chem. Commun. 1999, 261-
262.
[35] Li, W.; Zhang, Z.; Xiao, D.; Zhang, X. Tetrahedron Lett. 1999, 40 (37),
6701-6704.
[36] Li, W.; Zhang, X., J. Org. Chem. 2000, 65 (18), 5871-5874.
[37] Yan, Y.-Y.; RajanBabu, T. V. J. Org. Chem. 2000, 65 (3), 900-906.
[38] Yan, Y.-Y.; RajanBabu, T. V. Org. Lett. 2000, 2 (2), 199-202.
[39] Rajan Babu, T. V.; Yan, Y.-Y.; Shin, S. J. Am. Chem. Soc. 2001, 123
(42), 10207-10213.
INDEX
antisense, 9
# anuria, 46
aorta, 49
21st century, 59
apoptosis, 51, 54, 60
apoptotic mechanisms, 59
A appendicitis, 47
areolae, viii, 21, 23, 24, 25, 26, 27, 28, 29,
ABC transporters, vii, 1, 3 30, 31, 32, 33, 34, 35, 36, 37, 38
acetic acid, 44 arteries, 46
acid, 3, 4, 15, 25, 44, 55, 62, 71, 90, 92, 94, artery, 53
95, 98, 104, 105, 107 arthralgia, 47
acidic, viii, 41, 42, 90 arthrodesis, 47
acrylic acid, 107 arthroplasty, 47
active oxygen, 2 assessment, 50
active transport, 46 asthma, 50, 54
acute lung injury, 52, 59 atmosphere, 71
adaptation, 37 atoms, 106
additives, 45 ATP, 3, 4, 5
adenoma, 47 attachment, 12
adenopathy, 47
adjustment, 64
B
adults, 50, 57
age, 23, 47, 48, 49, 51
Bacillus subtilis, 8
alanine, 12, 14, 53
bacteria, vii, 1, 3, 4, 5, 6, 7, 14, 15, 16, 43
Alaska, 26, 39
bacterial cells, vii, 2, 3
algae, vii, viii, 1, 2, 12, 21, 22, 24, 28, 43
bacterium, 12, 13
alimentation, 1
BBB, 46
amino, 4, 6, 12, 13, 30, 60
behaviors, 72, 73
amino acid(s), 4, 6, 12, 13, 60
Bilateral, 47
ANOVA, 33
bile, 49, 55
antioxidant, 51, 52, 54, 59
114 Index
bile duct, 49 catalytic system, 105

bioavailability, 54 CCA, 14
biological fluids, 55, 61 CCR, 11, 13
biopsy, 47 cell biology, 39
biosynthesis, viii, 2, 43 cell death, 53
bleeding, 49 cell size, 51
blood, 46, 50, 52, 53, 56, 60, 61 cerebral edema, 46, 50, 56, 57
blood flow, 46 chemical, ix, 14, 16, 42, 44, 55, 63, 88
blood pressure, 50, 56 chemotherapy, 54, 61
blood vessels, 46 children, 57
blood-brain barrier, 56, 61 China, 87
bone, 47 chiral center, 92
brain, viii, 41, 46, 50, 51, 53, 54, 56, 58, 60, chirality, 98, 106, 108
61 cholecystectomy, 47
brain tumor, 50, 54, 58, 61 cholecystitis, 47
brainstem, 57 chromatography, viii, 21, 30, 39, 55, 62
bronchial epithelial cells, 52, 58 chronic heart failure, 56
bronchiectasis, 50, 53, 57 chronic obstructive pulmonary disease, 56
Brooks Range, 39 classification, 66
building blocks, 88 cleaning, 47
cleavage, 90
clinical disorders, vii, ix, 41, 46, 50, 51
C clusters, 24
CO2, 65
Ca2+, 61
coding, 8
cancer, 47
codon, 9
candidates, ix, 64, 71, 82, 83
color, iv
capillary, 30
coma, 50
carbohydrate, vii, viii, x, 1, 2, 3, 5, 6, 10, 21,
communities, 29, 39
22, 24, 26, 27, 28, 30, 32, 33, 34, 35, 37,
community, 69
39, 40, 87, 88
competition, 37
carbohydrate metabolism, 27, 28, 39
compounds, 60, 61
carbohydrates, vii, viii, 14, 16, 21, 27, 28,
condensation, 88
29, 30, 32, 33, 34, 35, 38, 39, 40
conditioning, ix, 63
carbon, vii, 1, 3, 11, 14, 15, 16, 43, 44, 105
conductivity, ix, 64, 83
carbon atoms, 105
configuration, 2, 91, 92, 105, 106
carbon dioxide, 44
congestive heart failure, 46
cardiopulmonary bypass, 46
conjugation, 109
carob, 15
containers, ix, 64
case studies, 84
contamination, 32
case study, 39
cooling, ix, 63, 65, 69, 70, 71, 74, 77, 78,
catabolism, 3, 57
82, 83, 84, 85
catalysis, x, 87, 88, 109
coordination, 88
catalyst, 15, 43, 95, 100, 104, 107, 108
COPD, 57
catalytic properties, 104
copper, 92
catalytic reduction, viii, 2
Index 115
correlation, 28, 30, 35, 58 distress, 47

correlation coefficient, 30 distribution, 28, 53, 60, 62, 89
cortex, 22, 24 diuretic, viii, 14, 41, 42, 56
cost, 65, 109 D-mannitol backbone, x, 87, 92
cough, 53 DNA, 9, 10, 11, 12
coughing, 50 drainage, 48
covering, 24 drug agents, viii, 41
craniotomy, 53 drugs, 46, 53, 54
crust, 22 DSC, 71, 72, 73, 74, 75, 81, 82, 85
crystal structure, 9 DSM, 5
crystalline, viii, 41, 42, 74, 78, 81
crystallization, 55
crystals, 45, 56 E
cuticle, 24
ecology, 37
cycles, 72, 74, 106
edema, 50, 57
cyst, 49
electricity, 65
cystectomy, 47
electron, 15
cysteine, 7, 8, 10, 12
emergency, 46, 51
cystic fibrosis, 50, 53, 56, 57, 59
encephalopathy, 48
cytochrome, 53
encoding, vii, 2, 3, 5, 8, 9, 10, 12, 14
cytoplasm, 3, 6, 7
endothelial cells, 46, 54
endothermic, ix, 63, 68
D energy, vii, ix, 1, 3, 4, 8, 16, 43, 46, 63, 64,
65, 66, 67, 68, 69, 76, 82, 84
damages, iv energy consumption, 77
database, 81 energy efficiency, 69
degradation, 51 energy supply, 65
degree of crystallinity, 54 energy transfer, 8
dehydration, 34, 88, 92 engineering, 55
dephosphorylation, 3, 16 environment, 2, 3, 9, 37, 40
deposition, 25 environmental impact, 65
depth, 24 environments, 34, 37
derivatives, 6, 30, 61, 104, 109 enzyme(s), vii, 2, 3, 4, 6, 15, 16, 52, 55
DHS, 40 eosinophilia, 50
diabetes, 52, 59 epinephrine, 54, 61
diarrhea, 48 epithelium, 52, 58, 59, 60
differential scanning, ix, 64, 71, 83, 84 equilibrium, 53
differential scanning calorimeter, 72 equipment, 72, 75, 81
differential scanning calorimetry, ix, 64, 71, ester, 98, 107
83, 84 ethanol, 30, 45
diffusion, 3, 46, 58 etherification, 92
discharges, 54 ethylene, 103
diseases, 48 Europe, 40
disorder, 51 evidence, 22, 25, 34, 37, 40
distilled water, 30 evolution, 16
116 Index
excretion, viii, 41, 46 granules, 37

experimental condition, 74 Great Britain, 40
exporter, 15 green alga, 24
exposure, 23, 53 growth, vii, viii, 2, 3, 15, 22, 25, 26, 27, 28,
extraction, 45 29, 32, 37, 38, 39, 40, 45, 56
extracts, 30, 38, 39, 55, 58 growth mechanism, 56
growth rate, viii, 22, 26, 32, 39, 40
F
H
fermentation, 43
fibroblasts, 52, 58 habitat, 12, 22, 26
fibrosis, 46, 50, 51, 53, 57 half-life, 51
filtration, 45 harvesting, 65
flexibility, 88, 92, 94, 109 head injury, 58
flora, 29 head trauma, 45, 52
fluid, viii, 41, 46, 69, 76, 82 heat capacity, 66
food, 14, 16, 42 heat transfer, 66, 69, 76, 82, 84
food industry, 16 heating rate, ix, 64, 71, 83
food products, 14 hemodialysis, 56
formation, 12, 25, 52, 75 hemoglobin, 52, 58
formula, 42 hepatic failure, 57
fragments, 105 hernia, 48
France, 1 hexane, 97
freedom, 100 histidine, 11, 12
fructose, vii, viii, 1, 2, 3, 4, 5, 6, 14, 15, 16, homogeneity, 8
31, 41, 42, 43, 88 human, 51, 52, 58, 59, 60, 62
FTIR, 74, 75 human brain, 60
functionalization, 109 human skin, 58
fungi, vii, 1, 3, 32, 39, 43, 44 hydrogen, 15, 52
fungus, viii, 21, 22, 27, 28, 43 hydrogen gas, 15
fusion, 13, 25, 48, 70, 76 hydrogenation, viii, x, 14, 15, 41, 42, 87, 88,
90, 93, 94, 95, 98, 99, 100, 102, 103,
104, 105, 107, 108, 109
G hydrolysis, 90
hydroquinone, 82
gangrene, 49
hydroxyl, 52, 88, 92, 98, 103, 104, 108
gastroenteritis, 48
hydroxyl groups, 89, 92, 98, 104
genes, vii, 2, 3, 4, 5, 8, 9, 12, 14, 15
hypertonic saline, 56, 57, 59
genus, 25, 28 hypertrophy, 48
geometry, 98 hysterectomy, 48
glucose, viii, 2, 8, 11, 12, 13, 15, 16, 31, 44,
52, 58
glutathione, 52, 56 I
glycerol, 3, 32
glycine, 53 ID, 17
Index 117
identification, 29, 39 liquids, 67

identity, 4, 12 Listeria monocytogenes, 8
in vitro, 8, 59, 60 liver, 50, 57
indirect measure, 39 liver failure, 50, 57
individuals, 25 localization, 5
induction, 3, 9, 11, 13, 101, 106 longevity, 23
inflammation, 50, 56, 57 lung function, 50
inhaler, 56 Luo, 57, 84, 110
initiation, 38, 56 lymphocytes, 59
injury, iv, 56, 57, 60
insulin, 52, 59
integration, 22 M
International Energy Agency (IEA), 65
macular degeneration, 51
intracranial pressure, viii, 41, 45, 50, 58
malaria, 50, 58
iodine, 29
maltose, 43
ion-driven co-transport systems, vii, 1
management, 45
IR spectroscopy, 75
marginal hypothallus, viii, 21
Ireland, 38, 40
Mars, 20
ischemia, 57, 59
mass, 55, 62, 67
isomers, 43, 54, 60
mass spectrometry, 55, 62
mastectomy, 49
K mastitis, 48
materials, ix, 63, 65, 66, 67, 68, 69, 70, 71,
Kaposi sarcoma, 49 76, 82, 83, 84, 104
ketones, 89 matrix, 12
kidney(s), 45, 46, 47, 50, 58, 60 matter, iv, 52, 58
measurement, 29, 39, 55, 61, 62
media, 3, 51, 94, 108
L medicine, 14, 16
medulla, 22, 29
lactate dehydrogenase, 15
medulloblastoma, 57
lactic acid, 3, 4, 5, 7, 13, 15, 16, 43 melt, ix, 64, 69
Lactobacillus, 7, 15
melting, ix, 64, 70, 73, 74, 78, 79, 81, 83
laparoscopy, 49
melting temperature, 70, 78
leaching, 38
membranes, 27
lead, 12, 13, 51, 106
meningitis, 50, 57
leakage, 26, 28, 32
Metabolic, 42, 44, 48
leucine, 53
metabolism, 3, 16, 39
liberation, 49 metabolized, 12
lichen, vii, viii, 1, 21, 22, 24, 25, 26, 28, 29, metal complexes, 95
32, 37, 38, 39, 40
metastasis, 47
ligand, 88, 93, 94, 95, 96, 97, 98, 99, 100,
methanol, 45, 108
101, 102, 103, 104, 105, 106, 107, 108
methyl group(s), 90, 98
liquid chromatography, 39, 55, 61
Mexico, 39, 41
liquid phase, 30
microcirculation, 56
118 Index
microorganisms, 22, 43 osmotic pressure, 2

microscope, 30 osmotic stress, 51, 53
mitochondria, 53 oxidation, 15, 102
modifications, 88 oxidative damage, 58
molasses, 15 oxidative stress, 51, 52, 58, 59
molecules, 43, 46 oxygen, 51
montelukast, 60
Moon, 60
motif, 9 P
MR, 58
pain, 47, 48, 54, 61
MRI, 58
palladium, 101, 106
mucus, 53, 56
pancreas, 52, 59
multiple regression, 30, 35, 36
pancreatitis, 52
multiple regression analysis, 35, 36
partition, 8, 55
mutant, 14
pathogens, 56
myocardial infarction, 48
pathways, vii, 1, 4, 43, 44
PCM, ix, 63, 66, 67, 69, 70, 71, 74, 76, 77,
N 78, 79, 82, 83, 84, 85
PEP, vii, 1, 3, 6, 7, 8, 11, 13
Na+, viii, 41, 46, 61 perfusion, 52, 54, 60
NAD, 15, 61 peripheral blood, 52
NADH, 3, 15 peripheral blood mononuclear cell, 52
nerve, 54, 61 permeability, 61, 62
neutral, 108 permeation, 61
nickel, 43 permission, iv
nitric oxide, 50, 57 peroxide, 51
NMR, 8 persistent asthma, 57
Norway, 26, 39 pH, viii, 41
nutrients, 22, 26, 28, 37 phagocytosis, 59
pharmaceutical(s), 45, 46, 53
phenylalanine, 105
O phlebotomy, 57
phosphate, 13, 43
obstruction, 49
phosphoenolpyruvate, vii, 1, 3
occlusion, 49
phosphorous, vii, x, 87, 105, 108
OECD, 64
phosphorus, 106
OH, 30, 42
phosphorylation, vii, 1, 6, 7, 8, 10, 11, 12,
oil, 65
13, 16, 51
olefins, 103 phosphotransferase system (PTS), vii, 1, 3
omeprazole, 54, 60 photosynthesis, viii, 21, 22, 37
operon, vii, 2, 3, 5, 6, 8, 9, 10, 11, 13, 14, 16
photovoltaic panels, 65
optimization, 16
Physiological, 38
organism, 7, 8, 9, 10, 15, 22, 24
physiology, 39, 40
organs, 54
pitch, 77
osmolality, 60
Index 119
plants, vii, viii, 1, 2, 16, 39, 40, 41, 43, 44, reconstruction, 49
65 recovery, 51, 60
platelets, 59 regenerate, 15
platform, 102 regeneration, 2, 15, 25
platinum, 60 renal dysfunction, 52
pneumonia, 49 renal failure, viii, 41, 45
polar, 34, 40 renewable energy, ix, 63, 65, 66, 82
pollution, 58 repression, vii, 2, 3, 13
polymer(s), 56, 88 repressor, 8, 9, 14
polymerase, 9 requirements, 69, 104
polymorphism, 85 researchers, 67
pools, 88 reserves, 27
population, 29, 51 residues, 10, 51
positive correlation, 35 resins, 30
power generation, 84 resistance, viii, 21, 34, 37, 59
preparation, iv, 88, 95 resources, 88
preservation, 60 response, 9, 10, 14, 54, 59, 71
prevention, 60 RH, 35
promoter, 9, 10, 11, 12 rhodium, 95, 100
proteasome, 51 rights, iv
protection, 16, 22, 27, 43, 54, 108 rings, 92, 101
proteins, 4, 5, 7, 8, 9, 10, 11, 12, 27, 53 risks, 67
prothallus, viii, 21, 22, 23, 24, 25, 26, 27, RNA, 9
28, 29, 30, 31, 32, 33, 34, 35, 36, 37 roots, 3, 61
purity, 88 routes, 3, 16
PVP, 45 rowing, 23
runoff, 26
Q
S
quantification, 30
savings, 69
secretion, 55
R selectivity, 108
sensitivity, 54, 60
radicals, 52
sensors, 77, 78
radiotherapy, 50, 57
sepsis, 49
radius, 49
serum, 61, 62
rainfall, 34
services, iv
RE, 38 shape, 24, 77, 78
reaction time, 93 shock, 47
reactions, 88, 98, 108
side effects, vii
reactive oxygen, 58, 59
signals, 46
reactivity, 105
signs, 51
receptors, 51, 57
skeletal muscle, 59
recommendations, iv
skeleton, 98
120 Index
skin, 52 surplus, 69
small intestine, 48 survival, 37
sodium, 46, 54, 60 susceptibility, 46, 51, 57
software, 30 swelling, 46, 50, 51, 58
solar collectors, ix, 64, 69, 70 Switzerland, 26, 40
solid state, 69 symbiosis, 22, 39
solidification, 74, 77, 79 symmetry, 2
solubility, 106 syndrome, 49, 54, 61
solution, 29, 45, 46, 65, 66, 69, 103 synthesis, x, 3, 12, 16, 28, 42, 43, 87, 88,
sorption, 84 90, 95, 98, 102, 106, 108
Spain, 63, 76
species, viii, 2, 13, 14, 22, 25, 28, 29, 32,
34, 37, 39, 58, 59 T
specific heat, ix, 64, 66, 67, 83
target, 11
spectroscopy, 8, 58
techniques, ix, 39, 41
speculation, 37
technologies, ix, 63, 82
sputum, 50
technology, ix, 63
stability, 51, 56
temperature, ix, 30, 34, 63, 66, 67, 68, 69,
starch, viii, 41, 42, 43, 53, 60, 88
70, 71, 74, 76, 77, 78, 79, 81, 82, 83, 84
state(s), ix, 11, 63, 66, 82, 84
tendon, 47, 49
stenosis, 48
testing, 62, 85
steroids, 47
tetrahydrofuran, 92
stimulation, 54
TGF, 46, 51
storage, vii, ix, 2, 43, 63, 65, 66, 67, 68, 69,
therapy, 50, 56, 58, 59
70, 77, 82, 83, 84, 85
thermal energy, vii, ix, 63, 65, 66, 70, 82,
stress, viii, 2, 16, 21, 27, 28, 34, 37, 43, 51,
83, 84, 85
55, 59
thermal energy storage (TES), ix, 63, 65
stretching, 53
thermal properties, 77, 83
structural changes, 8, 9
thermochemical materials (TCM), ix, 63
structure, 22, 37, 75, 88, 106
tissue, 23, 24, 25, 32, 33, 39, 46, 47
styrene, 95, 96
tobacco, 16
substitution, 101
toluene, 97, 105, 107
substrate(s), vii, 1, 3, 6, 9, 12, 93, 100, 104
transcription, 5, 8, 9, 10, 11, 12, 13, 14
success rate, 54
transformation, ix, 16, 43, 64, 83, 88
sucrose, 31, 43, 55, 88
transformations, 74
Sugar alcohols, 39
transforming growth factor, 46, 57
sugarcane, 15
transition metal, 88, 109
sulfate, 102
translocation, 26, 37
sulfur, 106
transplant, 30
sulfuric acid, 88
transport, vii, viii, 1, 3, 4, 5, 6, 8, 13, 14, 16,
Sun, 58
22, 28, 32, 46, 53
suppression, 60
transportation, 46
surface area, 29
traumatic brain injury, 50, 59
surface layer, 22
treatment, viii, 41, 45, 50, 51, 53, 54, 56, 57,
surgical resection, 50
58, 61
Index 121
trial, 57, 58, 59

tumor(s), 49, 50
W
type 1 diabetes, 59
Wales, viii, 21, 23, 26, 28, 29, 34, 37, 40
tyrosine, 13, 51
Washington, 26, 37
water, viii, 2, 34, 41, 42, 45, 46, 53, 54, 60,
U 61, 65, 66, 67, 69, 106, 107, 108
water absorption, 69
UK, viii, 21, 23, 26, 29, 30
urban, 58
urine, 55, 61, 62
X
USA, 19, 26, 30, 39, 84
X-ray diffraction (XRD), 80, 81
V
Y
vapor, ix, 64
yield, viii, 2, 15, 43, 45, 90, 92, 107, 108
variables, 35, 36
vasodilator, viii, 41, 42
vision, 51 Z
zoospore, 40

(Pharmacology-Research, Safety Testing and Regulation) Paolo Fubini-Mannitol - Chemistry, Uses and Potential Side Effects-Nova Science Pub Inc (2013)

Uploaded by

Document Information

Original Title

Copyright

Available Formats

Share this document

Share or Embed Document

Sharing Options

Did you find this document useful?

Is this content inappropriate?

Copyright:

Available Formats

(Pharmacology-Research, Safety Testing and Regulation) Paolo Fubini-Mannitol - Chemistry, Uses and Potential Side Effects-Nova Science Pub Inc (2013)

Uploaded by

Copyright:

Available Formats

PHARMACOLOGY - RESEARCH, SAFETY TESTING AND REGULATION

Additional books in this series can be found on Novas website

Additional e-books in this series can be found on Novas website

Additional books in this series can be found on Novas website

Additional e-books in this series can be found on Novas website

NOTICE TO THE READER

Independent verification should be sought for any data, advice or recommendations

Library of Congress Cataloging-in-Publication Data

Library of Congress Control Number: 2013945783

Published by Nova Science Publishers, Inc. New York

possible mechanisms of mannitol and its metabolite that are involved in

UTILIZATION AND PRODUCTION

Josef Deutscher1,2,3,*, Houda Bouraoui1,2,4,

transported by the PTS therefore arrives as D-mannitol-1-P in bacterial cells.

contain in the roots a mannose 1-oxidoreductase, which converts D-mannitol into

UPTAKE AND METABOLISM OF D-MANNITOL

of the family pseudomonadaceae, where the mtl operon is formed by the

Figure 2. The gene organization of D-mannitol-specific regions in different bacteria.

Corynebacterium glutamicum was reported to lack a fructokinase converting

other actinobacteria, such as Leifsonia xyli and Nakamurella multipartita as well

Figure 3. Schematic presentation of the different modes of organization of D-mannitol-

Interestingly, some firmicutes, such as Streptococcus mutans [21] or

enterococci, such as E. faecalis, Enterococcus faecium, Enterococcus

REGULATION OF MTL OPERON EXPRESSION

interaction of MtlR with the unphosphorylated EIIBMtl domain is also necessary to

PRODUCTION OF D-MANNITOL BY BACTERIA

free chewing gums. D-Mannitol is presently mainly produced by the chemical

[22] A. Maz, G. Bol, M. Ziga, A. Bourand, V. Loux, M. J. Yebra, V.

[41] L. M. Mustachio, S. Aksit, R. H. Mistry, R. Scheffler, A. Yamada and J. M.

Corresponding author: R.A. Armstrong Tel. +44-121-204-4102, Fax. +44-121-204-4048, Email.

concentrations of the other carbohydrates in the prothallus and not to their

Keywords: Lichen, symbiosis, Rhizocarpon geographicum, mannitol, prothallus,

(Figure 1) (Armstrong, 2011). This unusual organism comprises yellow-green

The areolae are highly variable in shape and morphological differences

Rhizocarpon geographicum poses several interesting biological problems.

Four processes contribute to the development of a mature thallus of R.

Rhizocarpon geographicum is one of the slowest growing of all crustose

Table 1. Examples of the reported growth rates (radial growth rate,

Location RGR (mm yr-1) Author

Field experiments have suggested that the prothallus of R. geographicum may

Soluble carbohydrate may account for up to 10% of thallus dry weight in

fungal prothallus is unknown in Rhizocarpon-type lichens. In some foliose and

A number of aspects of soluble carbohydrate metabolism may be important in

MATERIALS AND METHODS

Identification to species can be difficult in the yellow-green Rhizocarpon

Samples of areolae and prothallus were obtained from a population of R.

The concentrations of carbohydrates were determined by gas chromatography

A typical gas chromatogram trace obtained from a sample of areolae collected

The concentrations of ribitol, arabitol, and mannitol in the prothallus and

Table 2. Mean concentration (g g-1 extracted tissue, SEM in parentheses)

Carbohydrate Marginal prothallus Central areolae t

The soluble carbohydrates identified in the samples of R. geographicum were

There is some evidence of a change in the ratio of soluble carbohydrates in

CORRELATIONS BETWEEN CARBOHYDRATES

Table 3. Correlations (Pearsons r) between the concentrations of ribitol

The possible interrelationships between the three soluble carbohydrates in the

Table 4. Forward stepwise multiple regression analysis of the concentrations

Y variable X selected SSRED F SSEX R t

Armstrong RA. The biology of the crustose lichen Rhizocarpon geographicum.

MANNITOL: DISEASE-RELATED CHANGES

David Caldern Guzmn1, Gerardo Barragn Meja1,

biochemical markers used to monitor mannitol and its metabolite are

Table 1. Chemical properties of mannitol